Fungal resistant plants expressing casar

ABSTRACT

The present invention relates to a method of increasing resistance against fungal pathogens of the order Pucciniales, preferably the family Phacopsoraceae, in plants and/or plant cells. This is achieved by increasing the expression of a CASAR protein or fragment thereof in a plant, plant part and/or plant cell in comparison to wild type plants, wild type plant parts and/or wild type plant cells. Furthermore, the invention relates to transgenic plants, plant parts, and/or plant cells having an increased resistance against fungal pathogens, in particular, pathogens of the order Pucciniales, preferably the family Phacopsoraceae, and to recombinant expression vectors comprising a sequence that is identical or homologous to a sequence encoding a CASAR protein.

The applicant claims priority to EP 12192316.3 filed Nov. 13, 2012,which is incorporated herein by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention relates to a method of increasing resistanceagainst fungal pathogens, in particular, pathogens of the orderPucciniales, for example soybean rust, in plants, plant parts, and/orplant cells. This is achieved by increasing the expression and/oractivity of a CASAR protein in a plant, plant part and/or plant cell incomparison to wild type plants, wild type plant parts and/or wild typeplant cells.

Furthermore, the invention relates to transgenic plants, plant parts,and/or plant cells having an increased resistance against fungalpathogens, in particular, pathogens of the order Pucciniales, forexample soybean rust, and to recombinant expression vectors comprising asequence that is identical or homologous to a sequence encoding a CASARprotein.

BACKGROUND OF THE INVENTION

The cultivation of agricultural crop plants serves mainly for theproduction of foodstuffs for humans and animals. Monocultures inparticular, which are the rule nowadays, are highly susceptible to anepidemic-like spreading of diseases. The result is markedly reducedyields. To date, the pathogenic organisms have been controlled mainly byusing pesticides. Nowadays, the possibility of directly modifying thegenetic disposition of a plant or pathogen is also open to man.

Resistance generally describes the ability of a plant to prevent, or atleast curtail the infestation and colonization by a harmful pathogen.Different mechanisms can be discerned in the naturally occurringresistance, with which the plants fend off colonization byphytopathogenic organisms. These specific interactions between thepathogen and the host determine the course of infection (Schopfer andBrennicke (1999) Pflanzenphysiologie, Springer Verlag,Berlin-Heidelberg, Germany).

With regard to the race specific resistance, also called hostresistance, a differentiation is made between compatible andincompatible interactions. In the compatible interaction, an interactionoccurs between a virulent pathogen and a susceptible plant. The pathogensurvives, and may build up reproduction structures, while the hostmostly dies off. An incompatible interaction occurs on the other handwhen the pathogen infects the plant but is inhibited in its growthbefore or after weak development of symptoms (mostly by the presence ofR genes of the nucleotide-binding site-leucine-rich repeat (NBS-LRR)family, see below). In the latter case, the plant is resistant to therespective pathogen (Schopfer and Brennicke, vide supra). However, thistype of resistance is specific for a certain strain or pathogen.

In both compatible and incompatible interactions a defensive andspecific reaction of the host to the pathogen occurs. In nature,however, this resistance is often overcome because of the rapidevolutionary development of new virulent races of the pathogens (Neu etal. (2003) American Cytopathol. Society, MPMI 16 No. 7: 626-633).

Most pathogens are plant-species specific. This means that a pathogencan induce a disease in a certain plant species, but not in other plantspecies (Heath (2002) Can. J. Plant Pathol. 24: 259-264). The resistanceagainst a pathogen in certain plant species is called non-hostresistance. The non-host resistance offers strong, broad, and permanentprotection from phytopathogens. Genes providing non-host resistanceprovide the opportunity of a strong, broad and permanent protectionagainst certain diseases in non-host plants. In particular, such aresistance works for different strains of the pathogen.

Fungi are distributed worldwide. Approximately 100 000 different fungalspecies are known to date. Thereof rusts are of great importance. Theycan have a complicated development cycle with up to five different sporestages (spermatium, aecidiospore, uredospore, teleutospore andbasidiospore).

During the infection of plants by pathogenic fungi, different phases areusually observed. The first phases of the interaction betweenphytopathogenic fungi and their potential host plants are decisive forthe colonization of the plant by the fungus. During the first stage ofthe infection, the spores become attached to the surface of the plants,germinate, and the fungus penetrates the plant. Fungi may penetrate theplant via existing ports such as stomata, lenticels, hydatodes andwounds, or else they penetrate the plant epidermis directly as theresult of the mechanical force and with the aid of cell-wall-digestingenzymes. Specific infection structures are developed for penetration ofthe plant.

Immediately after recognition of a potential pathogen the plant startsto elicit defense reactions. Mostly the presence of the pathogen issensed via so called PAMP receptors, a class of trans-membrane receptorlike kinases recognizing conserved pathogen associated molecules (e.g.flagellin or chitin). Receptor-like kinases (RLKs) are signalingproteins that feature an extracellular domain connected via atransmembrane domain to a cytoplasmic kinase. This architectureindicates that RLKs perceive external signals, transducing them into thecell. In plants, RLKs were first implicated in the regulation ofdevelopment, in pathogen responses, and in recognition events. (SantiagoA Morillo and Frans E Tax (2006) Functional analysis of receptor-likekinases in monocots and dicots. Current Opinion in Plant Biology9:460-469).

Downstream of the PAMP receptors, the phytohormones salicylic acid (SA),jasmonate (JA) and ethylene (ET) play a critical role in the regulationof the different defense reactions. Depending on the ratio of thedifferent phytohormones, different defense reactions are elicited by thehost cell. Generally SA dependent defense is linked with resistanceagainst biotrophic pathogens, whereas JA/ET dependent defense reactionsare active against necrotrophic pathogens (and insects).

In addition to the localized defense responses, plants that wereattacked locally by a pathogen induce a “whole-plant” long lastingsystemic defence response called systemic acquired resistance (SAR). SARis associated with the induction of a wide range of genes (so called PRor “pathogenesis-related” genes), a burst of the reactive oxygen species(ROS), ethylene production and the accumulation of salicylic acid (SA).

Lee and Hwang (Planta 2005, Vol. 221: 790-800) showed that a gene calledSAR8.2 (also known as CASAR for “Capsicum annuum SAR”) accumulates insystemic pepper leaves as a result of a bacterial and fungal pathogeninfection, abiotic elicitors, and by environmental stresses such asdrought, salt, and low temperatures. The ectopic expression of CASAR inArabidopsis leads to a constitutive expression of the PR-genes includingAtPR-1, AtPR-4 and AtPR-5. Additionally the CASAR overexpression inArabidopsis enhanced the resistance against infections by Pseudomonassyringae pv. tomato, Fusarium oxysporum f.sp. matthiolae or Botrytiscinerea (Lee and Hwang, Plant Molecular Biology (2006) 61:95-109). Inaddition Lee and Hwang (Plant Molecular Biology (2006) 61:95-109) showedthat purified recombinant CASAR protein and crude protein extracts ofthe transgenic plants exhibited antifungal activity against somephytopathogenic fungi.

Lee and Hwang (Plant Molecular Biology (2006) 61:95-109) do notdemonstrate enhanced resistance or antifungal activity againstheminectrotrophic fungi, in particular against fungal pathogens of theorder Pucciniales (rust). This specific group of fungal pathogens ischaracterized by a unique life-cycle.

For instance, the soybean rust Phakopsora pachyrhizi directly penetratesthe plant epidermis. After crossing the epidermal cell, the fungusreaches the intercellular space of the mesophyll, where the fungusstarts to spread through the leaves. To acquire nutrients the funguspenetrates mesophyll cells and develops haustoria inside the mesophyllcell. During the penetration process the plasmamembrane of thepenetrated mesophyll cell stays intact. Therefore the soybean rustfungus establishes a biotrophic interaction with soybean.

The biotrophic phytopathogenic fungi, such as soybean rust and all otherrust fungi, depend for their nutrition on the metabolism of living cellsof the plants. This type of fungi belong to the group of biotrophicfungi, like other rust fungi, powdery mildew fungi or oomycete pathogenslike the genus Phytophthora or Peronospora The necrotrophicphytopathogenic fungi depend for their nutrition on dead cells of theplants, e.g. species from the genus Fusarium, Rhizoctonia orMycospaerella. Soybean rust has occupied an intermediate position, sinceit penetrates the epidermis directly, whereupon the penetrated cellbecomes necrotic.

After the penetration, the fungus changes over to anobligatory-biotrophic lifestyle. The subgroup of the biotrophic fungalpathogens which follows essentially such an infection strategy isheminecrotrophic. In contrast to a heminecrotrophic pathogen, ahemibiotrophic pathogen lives for a short period of time in a biotrophicmanner and subsequently starts killing the host cell and/or hostorganism, i.e., changes for the rest of its life-cycle to a necrotrophiclife-style.

Soybean rust has become increasingly important in recent times. Thedisease may be caused by the biotrophic rusts Phakopsora pachyrhizi andPhakopsora meibomiae. They belong to the class Basidiomycota, orderPucciniales (rust), previously also known as Uredinales, familyPhakopsoraceae. Both rusts infect a wide spectrum of leguminosic hostplants. P. pachyrhizi, also referred to as Asian rust, is the moreaggressive pathogen on soy (Glycine max), and is therefore, at leastcurrently, of great importance for agriculture. P. pachyrhizi can befound in nearly all tropical and subtropical soy growing regions of theworld. P. pachyrhizi is capable of infecting 31 species from 17 familiesof the Leguminosae under natural conditions and is capable of growing onfurther 60 species under controlled conditions (Sinclair et al. (eds.),Proceedings of the rust workshop (1995), National SoyaResearchLaboratory, Publication No. 1 (1996); Rytter J. L. et al., Plant Dis.87, 818 (1984)). P. meibomiae has been found in the Caribbean Basin andin Puerto Rico, and has not caused substantial damage as yet.

P. pachyrhizi can currently be controlled in the field only by means offungicides. Soy plants with resistance to the entire spectrum of theisolates are not available. When searching for resistant soybeanaccessions, six dominant R-genes of the NBS-LRR family, named Rpp1-5 andRpp? (Hyuuga), which mediate resistance of soy to P. pachyrhizi, werediscovered by screening thousands of soybean varieties. As the R-genesare derived from a host (soybean), the resistance was lost rapidly, asP. pachyrhizi develops new virulent races.

In recent years, fungal diseases, e.g. soybean rust, has gained inimportance as pest in agricultural production. There was therefore ademand in the prior art for developing methods to control fungi and toprovide fungal resistant plants.

Much research has been performed on the field of powdery and downymildew infecting the epidermal layer of plants. However, the problem tocope with soybean rust which infects the mesophyll remains unsolved.

The object of the present invention is inter alia to provide a method ofincreasing resistance against fungal pathogens, preferably rustpathogens (i.e., fungal pathogens of the order Pucciniales), preferablyagainst fungal pathogens of the family Phakopsoraceae, more preferablyagainst fungal pathogens of the genus Phakopsora, most preferablyagainst Phakopsora pachyrhizi and Phakopsora meibomiae, also known assoybean rust.

Surprisingly, we found that fungal pathogens, in particular of the orderPucciniales, in particular of the family Phakopsoraceae, for examplesoybean rust, can be controlled by increasing the expression of a CASARgene.

The present invention therefore provides a method of increasingresistance against fungal pathogens, preferably against rust pathogens(i.e., fungal pathogens of the order Pucciniales), preferably fungalpathogens of the family Phakopsoraceae, more preferably against fungalpathogens of the genus Phakopsora, most preferably against Phakopsorapachyrhizi and Phakopsora meibomiae, also known as soybean rust, intransgenic plants, transgenic plant parts, or transgenic plant cells byoverexpressing one or more CASAR nucleic acids.

A further object is to provide transgenic plants resistant againstfungal pathogens, preferably rust pathogens (i.e., fungal pathogens ofthe order Pucciniales), preferably of the family Phakopsoraceae, morepreferably against fungal pathogens of the genus Phakopsora, mostpreferably against Phakopsora pachyrhizi and Phakopsora meibomiae, alsoknown as soybean rust, a method for producing such plants as well as avector construct useful for the above methods.

Therefore, the present invention also refers to a recombinant vectorconstruct and a transgenic plant, transgenic plant part, or transgenicplant cell comprising an exogenous CASAR nucleic acid. Furthermore, amethod for the production of a transgenic plant, transgenic plant partor transgenic plant cell using the nucleic acid of the present inventionis claimed herein. In addition, the use of a nucleic acid or therecombinant vector of the present invention for the transformation of aplant, plant part, or plant cell is claimed herein.

The objects of the present invention, as outlined above, are achieved bythe subject-matter of the main claims. Preferred embodiments of theinvention are defined by the subject matter of the dependent claims.

BRIEF SUMMARY OF THE INVENTION

The object of the present invention is inter alia to provide a method ofincreasing resistance against fungal pathogens, preferably rustpathogens (i.e., fungal pathogens of the order Pucciniales), preferablyagainst fungal pathogens of the family Phakopsoraceae, more preferablyagainst fungal pathogens of the genus Phakopsora, most preferablyagainst Phakopsora pachyrhizi and Phakopsora meibomiae, also known assoybean rust.

Surprisingly, we found that resistance against fungal pathogens, inparticular of the family Phakopsoraceae, for example soybean rust, canbe enhanced by increasing the expression of a CASAR gene.

The present invention therefore provides a method of increasingresistance against fungal pathogens, preferably rust pathogens (i.e.,fungal pathogens of the order Pucciniales), preferably against fungalpathogens of the family Phakopsoraceae, more preferably against fungalpathogens of the genus Phakopsora, most preferably against Phakopsorapachyrhizi and Phakopsora meibomiae, also known as soybean rust, intransgenic plants, transgenic plant parts, or transgenic plant cells byoverexpressing one or more CASAR nucleic acids.

A further object is to provide transgenic plants resistant againstfungal pathogens, preferably rust pathogens (i.e., fungal pathogens ofthe order Pucciniales), preferably of the family Phakopsoraceae, morepreferably against fungal pathogens of the genus Phakopsora, mostpreferably against Phakopsora pachyrhizi and Phakopsora meibomiae, alsoknown as soybean rust, a method for producing such plants as well as avector construct useful for the above methods.

Therefore, the present invention also refers to a recombinant vectorconstruct and a transgenic plant, transgenic plant part, or transgenicplant cell comprising an exogenous CASAR nucleic acid. Furthermore, amethod for the production of a transgenic plant, transgenic plant partor transgenic plant cell using the nucleic acid of the present inventionis claimed herein. In addition, the use of a nucleic acid or therecombinant vector of the present invention for the transformation of aplant, plant part, or plant cell is claimed herein.

The objects of the present invention, as outlined above, are achieved bythe subject-matter of the main claims. Preferred embodiments of theinvention are defined by the subject matter of the dependent claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows the scoring system used to determine the level of diseasedleaf area of wildtype and transgenic soy plants against the rust fungusP. pachyrhizi (as described in GODOY, C. V., KOGA, L. J. & CANTERI, M.G. Diagrammatic scale for assessment of soybean rust severity.Fitopatologia Brasileira 31:063-068. 2006).

FIG. 2 shows the schematic illustration of the plant transformationvector harboring the CASAR cDNA under control of the parsley ubiquitinepromoter.

FIG. 3 shows the schematic illustration of the plant transformationvector harboring the CASAR cDNA under the control of the pathogeninduced “rust induced mesophyll specific promoter 820”.

FIG. 4 shows the full-length cDNA sequence of a CASAR gene from Capsicumannuum having SEQ ID NO: 1.

FIG. 5 shows the full-length cDNA sequence of a CASAR gene which has acodon optimization for optimal expression in soybean having SEQ ID NO:2.

FIG. 6 shows the sequence of a CASAR protein (SEQ ID NO: 3).

FIG. 7 shows the sequence of the rust induced mesophyll specificpromoter 820 having SEQ ID NO: 4.

FIG. 8 shows the result of the scoring of 43 transgenic soy plants(derived from 5 independent events) expressing the CASAR overexpressionvector construct. T₁ soybean plants expressing CASAR protein wereinoculated with spores of Phakopsora pachyrhizi. The evaluation of thediseased leaf area on all leaves was performed 14 days afterinoculation. The average of the percentage of the leaf area showingfungal colonies or strong yellowing/browning on all leaves wasconsidered as diseased leaf area. All 43 soybean T₁ plants expressingCASAR (expression checked by RT-PCR) were evaluated in parallel tonon-transgenic control plants. The average of the diseased leaf area isshown in FIG. 7. Overexpression of CASAR significantly (***: p<0.001)reduces the diseased leaf area in comparison to non-transgenic controlplants by 44.5%.

FIG. 9 contains a brief description of the sequences of the sequencelisting.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the examples included herein.

DEFINITIONS

Unless otherwise noted, the terms used herein are to be understoodaccording to conventional usage by those of ordinary skill in therelevant art. In addition to the definitions of terms provided herein,definitions of common terms in molecular biology may also be found inRieger et al., 1991 Glossary of genetics: classical and molecular, 5thEd., Berlin: Springer-Verlag; and in Current Protocols in MolecularBiology, F. M. Ausubel et al., Eds., Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(1998 Supplement).

It is to be understood that as used in the specification and in theclaims, “a” or “an” can mean one or more, depending upon the context inwhich it is used. Thus, for example, reference to “a cell” can mean thatat least one cell can be utilized. It is to be understood that theterminology used herein is for the purpose of describing specificembodiments only and is not intended to be limiting.

Throughout this application, various publications are referenced. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains. Standard techniquesfor cloning, DNA isolation, amplification and purification, forenzymatic reactions involving DNA ligase, DNA polymerase, restrictionendonucleases and the like, and various separation techniques are thoseknown and commonly employed by those skilled in the art. A number ofstandard techniques are described in Sambrook et al., 1989 MolecularCloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.;Maniatis et al., 1982 Molecular Cloning, Cold Spring Harbor Laboratory,Plainview, N.Y.; Wu (Ed.) 1993 Meth. Enzymol. 218, Part I; Wu (Ed.) 1979Meth Enzymol. 68; Wu et al., (Eds.) 1983 Meth. Enzymol. 100 and 101;Grossman and Moldave (Eds.) 1980 Meth. Enzymol. 65; Miller (Ed.) 1972Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.; Old and Primrose, 1981 Principles of GeneManipulation, University of California Press, Berkeley; Schleif andWensink, 1982 Practical Methods in Molecular Biology; Glover (Ed.) 1985DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins(Eds.) 1985 Nucleic Acid Hybridization, IRL Press, Oxford, UK; andSetlow and Hollaender 1979 Genetic Engineering: Principles and Methods,Vols. 1-4, Plenum Press, New York. Abbreviations and nomenclature, whereemployed, are deemed standard in the field and commonly used inprofessional journals such as those cited herein.

“Homologues” of a protein encompass peptides, oligopeptides,polypeptides, proteins and/or enzymes having amino acid substitutions,deletions and/or insertions relative to the unmodified protein inquestion and having similar functional activity as the unmodifiedprotein from which they are derived.

“Homologues” of a nucleic acid encompass nucleotides and/orpolynucleotides having nucleic acid substitutions, deletions and/orinsertions relative to the unmodified nucleic acid in question, whereinthe protein coded by such nucleic acids has similar functional activityas the unmodified protein coded by the unmodified nucleic acid fromwhich they are derived. In particular, homologues of a nucleic acid mayencompass substitutions on the basis of the degenerative amino acidcode.

The terms “identity”, “homology” and “similarity” are used hereininterchangeably. “Identity” or “homology” or “similarity” between twonucleic acids sequences or amino acid sequences refers in each case overthe entire length of the respective CASAR nucleic acid sequence or CASARamino acid sequence.

Preferably, “percentage of sequence identity” is calculated by comparingtwo optimally aligned sequences over a particular region, determiningthe number of positions at which the identical base or amino acid occursin both sequences in order to yield the number of matched positions,dividing the number of such positions by the total number of positionsin the region being compared and multiplying the result by 100.

Methods for the alignment of sequences for comparison are well known inthe art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAPuses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48:443-453) to find the global (i.e. spanning the complete sequences)alignment of two sequences that maximizes the number of matches andminimizes the number of gaps. The BLAST algorithm (Altschul et al.(1990) J Mol Biol 215: 403-10) calculates percent sequence identity orsimilarity or homology and performs a statistical analysis of theidentity or similarity or homology between the two sequences. Thesoftware for performing BLAST analysis is publicly available through theNational Centre for Biotechnology Information (NCBI). Homologues mayreadily be identified using, for example, the ClustalW multiple sequencealignment algorithm (version 1.83), with the default pairwise alignmentparameters, and a scoring method in percentage. Global percentages ofsimilarity/homology/identity may also be determined using one of themethods available in the MatGAT software package (Campanella et al., BMCBioinformatics. 2003 Jul. 10; 4:29. MatGAT: an application thatgenerates similarity/homology/identity matrices using protein or DNAsequences.). Minor manual editing may be performed to optimise alignmentbetween conserved motifs, as would be apparent to a person skilled inthe art. Furthermore, instead of using full-length sequences for theidentification of homologues, specific domains may also be used. Thesequence identity values may be determined over the entire nucleic acidor amino acid sequence or over selected domains or conserved motif(s),using the programs mentioned above using the default parameters. Forlocal alignments, the Smith-Waterman algorithm is particularly useful(Smith T F, Waterman M S (1981) J. Mol. Biol 147(1); 195-7).

The sequence identity may also be calculated by means of the Vector NTISuite 7.1 program of the company Informax (USA) employing the ClustalMethod (Higgins D G, Sharp P M. Fast and sensitive multiple sequencealignments on a microcomputer. Comput Appl. Biosci. 1989 April;5(2):151-1) with the following settings:

Multiple Alignment Parameter:

Gap opening penalty 10 Gap extension penalty 10 Gap separation penaltyrange 8 Gap separation penalty off % identity for alignment delay 40Residue specific gaps off Hydrophilic residue gap off Transitionweighing 0

Pairwise Alignment Parameter:

FAST algorithm on K-tuple size 1 Gap penalty 3 Window size 5 Number ofbest diagonals 5

Alternatively the identity may be determined according to Chenna, Ramu,Sugawara, Hideaki, Koike, Tadashi, Lopez, Rodrigo, Gibson, Toby J,Higgins, Desmond G, Thompson, Julie D. Multiple sequence alignment withthe Clustal series of programs. (2003) Nucleic Acids Res 31(13):3497-500, the web page:http://www.ebi.ac.uk/Tools/clustalw/index.html# and the followingsettings

DNA Gap Open Penalty 15.0 DNA Gap Extension Penalty 6.66 DNA MatrixIdentity Protein Gap Open Penalty 10.0 Protein Gap Extension Penalty 0.2Protein matrix Gonnet Protein/DNA ENDGAP −1 Protein/DNA GAPDIST 4

Sequence identity between the nucleic acid or protein useful accordingto the present invention and the CASAR nucleic acids or CASAR proteinsmay be optimized by sequence comparison and alignment algorithms knownin the art (see Gribskov and Devereux, Sequence Analysis Primer,Stockton Press, 1991, and references cited therein) and calculating thepercent difference between the nucleotide or protein sequences by, forexample, the Smith-Waterman algorithm as implemented in the BESTFITsoftware program using default parameters (e.g., University of WisconsinGenetic Computing Group).

A “deletion” refers to removal of one or more amino acids from a proteinor to the removal of one or more nucleic acids from DNA, ssRNA and/ordsRNA.

An “insertion” refers to one or more amino acid residues or nucleic acidresidues being introduced into a predetermined site in a protein or thenucleic acid.

A “substitution” refers to replacement of amino acids of the proteinwith other amino acids having similar properties (such as similarhydrophobicity, hydrophilicity, antigenicity, propensity to form orbreak α-helical structures or beta-sheet structures).

On the nucleic acid level a substitution refers to a replacement of oneor more nucleotides with other nucleotides within a nucleic acid,wherein the protein coded by the modified nucleic acid has a similarfunction. In particular homologues of a nucleic acid encompasssubstitutions on the basis of the degenerative amino acid code.

Amino acid substitutions are typically of single residues, but may beclustered depending upon functional constraints placed upon the proteinand may range from 1 to 10 amino acids; insertions or deletion willusually be of the order of about 1 to 10 amino acid residues. The aminoacid substitutions are preferably conservative amino acid substitutions.Conservative substitution tables are well known in the art (see forexample Creighton (1984) Proteins. W.H. Freeman and Company (Eds) andTable 1 below or Taylor W. R. (1986) The classification of amino acidconservation J Theor Biol., 119:205-18).

TABLE 1 Examples of conserved amino acid substitutions ConservativeResidue Substitutions Ala Ser Arg Lys Asn Gln; His Asp Glu Gln Asn CysSer Glu Asp Gly Pro His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; GlnMet Leu; Ile Phe Met; Leu; Tyr Ser Thr; Gly Thr Ser; Val Trp Tyr TyrTrp; Phe Val Ile; Leu

Amino acid substitutions, deletions and/or insertions may readily bemade using peptide synthetic techniques well known in the art, such assolid phase peptide synthesis and the like, or by recombinant DNAmanipulation.

Methods for the manipulation of DNA sequences to produce substitution,insertion or deletion variants of a protein are well known in the art.For example, techniques for making substitution mutations atpredetermined sites in DNA are well known to those skilled in the artand include M13 mutagenesis, T7-Gene in vitro mutagenesis (USB,Cleveland, Ohio), QuickChange Site Directed mutagenesis (Stratagene, SanDiego, Calif.), PCR-mediated site-directed mutagenesis or othersite-directed mutagenesis protocols.

Orthologues and paralogues encompass evolutionary concepts used todescribe the ancestral relationships of genes. Paralogues are geneswithin the same species that have originated through duplication of anancestral gene; orthologues are genes from different organisms that haveoriginated through speciation, and are also derived from a commonancestral gene.

The terms “encode” or “coding for” is used for the capability of anucleic acid to contain the information for the amino acid sequence of aprotein via the genetic code, i.e., the succession of codons each beinga sequence of three nucleotides, which specify which amino acid will beadded next during protein synthesis. The terms “encode” or “coding for”therefore includes all possible reading frames of a nucleic acid.Furthermore, the terms “encode” or “coding for” also applies to anucleic acid, which coding sequence is interrupted by noncoding nucleicacid sequences, which are removed prior translation, e.g., a nucleicacid sequence comprising introns.

The term “domain” refers to a set of amino acids conserved at specificpositions along an alignment of sequences of evolutionarily relatedproteins. While amino acids at other positions can vary betweenhomologues, amino acids that are highly conserved at specific positionsindicate amino acids that are likely essential in the structure,stability or function of a protein.

Specialist databases exist for the identification of domains, forexample, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95,5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244),InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318), Prosite(Bucher and Bairoch (1994), A generalized profile syntax forbiomolecular sequences motifs and its function in automatic sequenceinterpretation. (In) ISMB-94; Proceedings 2nd International Conferenceon Intelligent Systems for Molecular Biology. Altman R., Brutlag D.,Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAI Press, Menlo Park;Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)), or Pfam (Batemanet al., Nucleic Acids Research 30(1): 276-280 (2002)). A set of toolsfor in silico analysis of protein sequences is available on the ExPASyproteomics server (Swiss Institute of Bioinformatics (Gasteiger et al.,ExPASy: the proteomics server for in-depth protein knowledge andanalysis, Nucleic Acids Res. 31:3784-3788(2003)). Domains or motifs mayalso be identified using routine techniques, such as by sequencealignment.

As used herein the terms “fungal-resistance”, “resistant to a fungus”and/or “fungal-resistant” mean reducing, preventing, or delaying aninfection by fungi. The term “resistance” refers to fungal resistance.Resistance does not imply that the plant necessarily has 100% resistanceto infection. In preferred embodiments, enhancing or increasing fungalresistance means that resistance in a resistant plant is greater than10%, greater than 20%, greater than 30%, greater than 40%, greater than50%, greater than 60%, greater than 70%, greater than 80%, greater than90%, or greater than 95% in comparison to a wild type plant.

As used herein the terms “soybean rust-resistance”, “resistant to asoybean rust”, “soybean rust-resistant”, “rust-resistance”, “resistantto a rust”, or “rust-resistant” mean reducing or preventing or delayingan infection of a plant, plant part, or plant cell by Phakopsoracea, inparticular Phakopsora pachyrhizi and Phakopsora meibomiae—also known assoybean rust or Asian Soybean Rust (ASR), as compared to a wild typeplant, wild type plant part, or wild type plant cell. Resistance doesnot imply that the plant necessarily has 100% resistance to infection.In preferred embodiments, enhancing or increasing rust resistance meansthat rust resistance in a resistant plant is greater than 10%, greaterthan 20%, greater than 30%, greater than 40%, greater than 50%, greaterthan 60%, greater than 70%, greater than 80%, greater than 90%, orgreater than 95% in comparison to a wild type plant that is notresistant to soybean rust. Preferably the wild type plant is a plant ofa similar, more preferably identical, genotype as the plant havingincreased resistance to the soybean rust, but does not comprise anexogenous CASAR nucleic acid, functional fragments thereof and/or anexogenous nucleic acid capable of hybridizing with a CASAR nucleic acid.

The level of fungal resistance of a plant can be determined in variousways, e.g. by scoring/measuring the infected leaf area in relation tothe overall leaf area. Another possibility to determine the level ofresistance is to count the number of soybean rust colonies on the plantor to measure the amount of spores produced by these colonies. Anotherway to resolve the degree of fungal infestation is to specificallymeasure the amount of rust DNA by quantitative (q) PCR. Specific probesand primer sequences for most fungal pathogens are available in theliterature (Frederick R D, Snyder C L, Peterson G L, et al. 2002Polymerase chain reaction assays for the detection and discrimination ofthe rust pathogens Phakopsora pachyrhizi and P. meibomiae,Phytopathology 92(2) 217-227).

The term “hybridization” as used herein includes “any process by which astrand of nucleic acid molecule joins with a complementary strandthrough base pairing” (J. Coombs (1994) Dictionary of Biotechnology,Stockton Press, New York). Hybridization and the strength ofhybridization (i.e., the strength of the association between the nucleicacid molecules) is impacted by such factors as the degree ofcomplementarity between the nucleic acid molecules, stringency of theconditions involved, the Tm of the formed hybrid, and the G:C ratiowithin the nucleic acid molecules.

As used herein, the term “Tm” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the Tm ofnucleic acid molecules is well known in the art. As indicated bystandard references, a simple estimate of the Tm value may be calculatedby the equation: Tm=81.5+0.41(% G+C), when a nucleic acid molecule is inaqueous solution at 1 M NaCl (see e.g., Anderson and Young, QuantitativeFilter Hybridization, in Nucleic Acid Hybridization (1985). Otherreferences include more sophisticated computations, which takestructural as well as sequence characteristics into account for thecalculation of Tm. Stringent conditions, are known to those skilled inthe art and can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

In particular, the term “stringency conditions” refers to conditions,wherein 100 contiguous nucleotides or more, 150 contiguous nucleotidesor more, 200 contiguous nucleotides or more or 250 contiguousnucleotides or more which are a fragment or identical to thecomplementary nucleic acid molecule (DNA, RNA, ssDNA or ssRNA)hybridizes under conditions equivalent to hybridization in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in2×SSC, 0.1% SDS at 50° C. or 65° C., preferably at 65° C., with aspecific nucleic acid molecule (DNA; RNA, ssDNA or ss RNA). Preferably,the hybridizing conditions are equivalent to hybridization in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in1×SSC, 0.1% SDS at 50° C. or 65° C., preferably 65° C., more preferablythe hybridizing conditions are equivalent to hybridization in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in0.1×SSC, 0.1% SDS at 50° C. or 65° C., preferably 65° C. Preferably, thecomplementary nucleotides hybridize with a fragment or the whole CASARnucleic acids. Alternatively, preferred hybridization conditionsencompass hybridisation at 65° C. in 1×SSC or at 42° C. in 1×SSC and 50%formamide, followed by washing at 65° C. in 0.3×SSC or hybridisation at50° C. in 4×SSC or at 40° C. in 6×SSC and 50% formamide, followed bywashing at 50° C. in 2×SSC. Further preferred hybridization conditionsare 0.1% SDS, 0.1 SSD and 65° C.

The term “plant” is intended to encompass plants at any stage ofmaturity or development, as well as any tissues or organs (plant parts)taken or derived from any such plant unless otherwise clearly indicatedby context. Plant parts include, but are not limited to, plant cells,stems, roots, flowers, ovules, stamens, seeds, leaves, embryos,meristematic regions, callus tissue, anther cultures, gametophytes,sporophytes, pollen, microspores, protoplasts, hairy root cultures,and/or the like. The present invention also includes seeds produced bythe plants of the present invention. Preferably, the seeds comprise theexogenous CASAR nucleic acids. In one embodiment, the seeds can developinto plants with increased resistance to fungal infection as compared toa wild-type variety of the plant seed. As used herein, a “plant cell”includes, but is not limited to, a protoplast, gamete producing cell,and a cell that regenerates into a whole plant. Tissue culture ofvarious tissues of plants and regeneration of plants therefrom is wellknown in the art and is widely published.

Reference herein to an “endogenous” nucleic acid and/or protein refersto the nucleic acid and/or protein in question as found in a plant inits natural form (i.e., without there being any human intervention).

The term “exogenous” nucleic acid refers to a nucleic acid that has beenintroduced in a plant by means of gene technology. An “exogenous”nucleic acid can either not occur in a plant in its natural form, bedifferent from the nucleic acid in question as found in a plant in itsnatural form, or can be identical to a nucleic acid found in a plant inits natural form, but integrated not within its natural geneticenvironment. The corresponding meaning of “exogenous” is applied in thecontext of protein expression. For example, a transgenic plantcontaining a transgene, i.e., an exogenous nucleic acid, may, whencompared to the expression of the endogenous gene, encounter asubstantial increase of the expression of the respective gene or proteinin total. A transgenic plant according to the present invention includesan exogenous CASAR nucleic acid integrated at any genetic loci andoptionally the plant may also include the endogenous gene within thenatural genetic background.

For the purposes of the invention, “recombinant” means with regard to,for example, a nucleic acid sequence, a nucleic acid molecule, anexpression cassette or a vector construct comprising any one or moreCASAR nucleic acids, all those constructions brought about by man bygene technological methods in which either

-   (a) the sequences of the one or more CASAR nucleic acid or a part    thereof, or-   (b) genetic control sequence(s) which is operably linked with the    CASAR nucleic acid sequence according to the invention, for example    a promoter, or-   (c) a) and b)    are not located in their natural genetic environment or have been    modified by man by gene technological methods. The modification may    take the form of, for example, a substitution, addition, deletion,    inversion or insertion of one or more nucleotide residues. The    natural genetic environment is understood as meaning the natural    genomic or chromosomal locus in the original plant or the presence    in a genomic library or the combination with the natural promoter.

For instance, a naturally occurring expression cassette—for example thenaturally occurring combination of the natural promoter of the nucleicacid sequences with the corresponding nucleic acid sequence encoding aprotein useful in the methods of the present invention, as definedabove—becomes a recombinant expression cassette when this expressioncassette is modified by man by non-natural, synthetic (“artificial”)methods such as, for example, mutagenic treatment. Suitable methods aredescribed, for example, in U.S. Pat. No. 5,565,350, WO 00/15815 orUS200405323. Furthermore, a naturally occurring expression cassette—forexample the naturally occurring combination of the natural promoter ofthe nucleic acid sequences with the corresponding nucleic acid sequenceencoding a protein useful in the methods of the present invention, asdefined above—becomes a recombinant expression cassette when thisexpression cassette is not integrated in the natural genetic environmentbut in a different genetic environment.

The term “isolated nucleic acid” or “isolated protein” refers to anucleic acid or protein that is not located in its natural environment,in particular its natural cellular environment. Thus, an isolatednucleic acid or isolated protein is essentially separated from othercomponents of its natural environment. However, the skilled person inthe art is aware that preparations of an isolated nucleic acid or anisolated protein can display a certain degree of impurity depending onthe isolation procedure used. Methods for purifying nucleic acids andproteins are well known in the art. The isolated gene may be isolatedfrom an organism or may be manmade, for example by chemical synthesis.In this regard, a recombinant nucleic acid may also be in an isolatedform.

As used herein, the term “transgenic” refers to an organism, e.g., aplant, plant cell, callus, plant tissue, or plant part that exogenouslycontains the nucleic acid, recombinant construct, vector or expressioncassette described herein or a part thereof which is preferablyintroduced by non-essentially biological processes, preferably byAgrobacteria transformation. The recombinant construct or a part thereofis stably integrated into a chromosome, so that it is passed on tosuccessive generations by clonal propagation, vegetative propagation orsexual propagation. Preferred successive generations are transgenic too.Essentially biological processes may be crossing of plants and/ornatural recombination.

A transgenic plant, plants cell or tissue for the purposes of theinvention is thus understood as meaning that an exogenous CASAR nucleicacid, recombinant construct, vector or expression cassette including oneor more CASAR nucleic acids is integrated into the genome by means ofgene technology.

A “wild type” plant, “wild type” plant part, or “wild type” plant cellmeans that said plant, plant part, or plant cell does not expressexogenous CASAR nucleic acid or exogenous CASAR protein.

Natural locus means the location on a specific chromosome, preferablythe location between certain genes, more preferably the same sequencebackground as in the original plant which is transformed.

Preferably, the transgenic plant, plant cell or tissue thereof expressesthe CASAR nucleic acids, CASAR constructs or CASAR expression cassettesdescribed herein.

The term “expression” or “gene expression” means the transcription of aspecific gene or specific genes or specific genetic vector construct.The term “expression” or “gene expression” in particular means thetranscription of a gene or genes or genetic vector construct intostructural RNA (rRNA, tRNA), or mRNA with or without subsequenttranslation of the latter into a protein. The process includestranscription of DNA and processing of the resulting RNA product. Theterm “expression” or “gene expression” can also include the translationof the mRNA and therewith the synthesis of the encoded protein, i.e.,protein expression.

The term “increased expression” or “enhanced expression” or“overexpression” or “increase of content” as used herein means any formof expression that is additional to the original wild-type expressionlevel. For the purposes of this invention, the original wild-typeexpression level might also be zero (absence of expression).

Methods for increasing expression of genes or gene products are welldocumented in the art and include, for example, overexpression driven byappropriate promoters, the use of transcription enhancers or translationenhancers. Isolated nucleic acids which serve as promoter or enhancerelements may be introduced in an appropriate position (typicallyupstream) of a non-heterologous form of a polynucleotide so as toupregulate expression of a nucleic acid encoding the protein ofinterest. For example, endogenous promoters may be altered in vivo bymutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No.5,565,350; Zarling et al., WO9322443), or isolated promoters may beintroduced into a plant cell in the proper orientation and distance froma gene of the present invention so as to control the expression of thegene.

The term “functional fragment” refers to any nucleic acid or proteinwhich comprises merely a part of the fulllength nucleic acid orfulllength protein, respectively, but still provides the same function,e.g., fungal resistance, when expressed or repressed in a plant,respectively. Preferably, the fragment comprises at least 50%, at least60%, at least 70%, at least 80%, at least 90% at least 95%, at least98%, at least 99% of the original sequence. Preferably, the functionalfragment comprises contiguous nucleic acids or amino acids as in theoriginal nucleic acid or original protein, respectively. In oneembodiment the fragment of any of the CASAR nucleic acids has anidentity as defined above over a length of at least 20%, at least 30%,at least 50%, at least 75%, at least 90% of the nucleotides of therespective CASAR nucleic acid.

The term “splice variant” as used herein encompasses variants of anucleic acid sequence in which selected introns and/or exons have beenexcised, replaced, displaced or added, or in which introns have beenshortened or lengthened. Thus, a splice variant can have one or more oreven all introns removed or added. According to this definition, a cDNAis considered as a splice variant of the respective intron-containinggenomic sequence and vice versa. Such splice variants may be found innature or may be manmade. Methods for predicting and isolating suchsplice variants are well known in the art (see for example Foissac andSchiex (2005) BMC Bioinformatics 6: 25).

In cases where overexpression of nucleic acid is desired, the term“similar functional activity” or “similar function” means that anyhomologue and/or fragment provide fungal resistance when expressed in aplant. Preferably similar functional activity means at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98%, at least 99% or 100% or higher fungal resistance comparedwith functional activity provided by the exogenous expression of theCASAR nucleotide sequence as defined by SEQ ID NO: 1 or 2 or the CASARprotein sequence as defined by SEQ ID NO: 3.

The term “increased activity” or “enhanced activity” as used hereinmeans any protein having increased activity and which provides anincreased fungal resistance compared with the wildtype plant merelyexpressing the respective endogenous CASAR nucleic acid. As far asoverexpression is concerned, for the purposes of this invention, theoriginal wild-type expression level might also be zero (absence ofexpression).

With respect to a vector construct and/or the recombinant nucleic acidmolecules, the term “operatively linked” is intended to mean that thenucleic acid to be expressed is linked to the regulatory sequence,including promoters, terminators, enhancers and/or other expressioncontrol elements (e.g., polyadenylation signals), in a manner whichallows for expression of the nucleic acid (e.g., in a host plant cellwhen the vector is introduced into the host plant cell). Such regulatorysequences are described, for example, in Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990) and Gruber and Crosby, in: Methods in Plant Molecular Biology andBiotechnology, Eds. Glick and Thompson, Chapter 7, 89-108, CRC Press:Boca Raton, Fla., including the references therein. Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cells and those that direct expression ofthe nucleotide sequence only in certain host cells or under certainconditions. It will be appreciated by those skilled in the art that thedesign of the vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of nucleic aciddesired, and the like.

The term “introduction” or “transformation” as referred to hereinencompass the transfer of an exogenous polynucleotide into a host cell,irrespective of the method used for transfer. Plant tissue capable ofsubsequent clonal propagation, whether by organogenesis orembryogenesis, may be transformed with a vector construct of the presentinvention and a whole plant regenerated there from. The particulartissue chosen will vary depending on the clonal propagation systemsavailable for, and best suited to, the particular species beingtransformed. Exemplary tissue targets include leaf disks, pollen,embryos, cotyledons, hypocotyls, megagametophytes, callus tissue,existing meristematic tissue (e.g., apical meristem, axillary buds, androot meristems), and induced meristem tissue (e.g., cotyledon meristemand hypocotyl meristem). The polynucleotide may be transiently or stablyintroduced into a host cell and may be maintained non-integrated, forexample, as a plasmid. Alternatively, it may be integrated into the hostgenome. The host genome includes the nucleic acid contained in thenucleus as well as the nucleic acid contained in the plastids, e.g.,chloroplasts, and/or mitochondria. The resulting transformed plant cellmay then be used to regenerate a transformed plant in a manner known topersons skilled in the art.

The term “terminator” encompasses a control sequence which is a DNAsequence at the end of a transcriptional unit which signals 3′processing and polyadenylation of a primary transcript and terminationof transcription. The terminator can be derived from the natural gene,from a variety of other plant genes, or from T-DNA. The terminator to beadded may be derived from, for example, the nopaline synthase oroctopine synthase genes, or alternatively from another plant gene, orless preferably from any other eukaryotic gene.

DETAILED DESCRIPTION CASAR Nucleic Acids

The CASAR nucleic acid to be overexpressed in order to achieve increasedresistance to fungal pathogens, e.g., of the family Phakopsoraceae, forexample soybean rust, is preferably a nucleic acid coding for a CASARprotein, and is preferably as defined by SEQ ID NO: 2, 1, 5-12, 13, 15,17, 19, 21, 23, 25, or 27, or a fragment, homolog, derivative,orthologue or paralogue thereof, or a splice variant thereof.Preferably, the nucleic acid coding for a CASAR protein of the presentinvention has at least 60% identity, preferably at least 70% sequenceidentity, at least 80%, at least 90%, at least 95%, at least 98%, atleast 99% sequence identity, or even 100% sequence identity with SEQ IDNO: 2, 1, 5-12, 13, 15, 17, 19, 21, 23, 25, or 27, or is a functionalfragment thereof, or a splice variant thereof. Most preferred is atleast 90% identity, at least 95% identity, more preferred is at least98% or at least 99% identity with SEQ ID NO: 2, 1, 5-12, 13, 15, 17, 19,21, 23, 25, or 27.

Preferably, the CASAR nucleic acid to be overexpressed in order toachieve increased resistance to fungal pathogens, e.g., of the familyPhakopsoraceae, for example soybean rust, is preferably a nucleic acidcoding for a CASAR protein, and is preferably as defined by SEQ ID NO:1, or a fragment, homolog, derivative, orthologue or paralogue thereof,or a splice variant thereof. Preferably, the nucleic acid coding for aCASAR protein of the present invention has at least 60% identity,preferably at least 70% sequence identity, at least 80%, at least 90%,at least 95%, at least 98%, at least 99% sequence identity, or even 100%sequence identity with SEQ ID NO: 1 or is a functional fragment thereof,or a splice variant thereof. Most preferred is at least 90% identity, atleast 95% identity, more preferred is at least 98% or at least 99%identity with SEQ ID NO: 1.

More preferably, the CASAR nucleic acid to be overexpressed in order toachieve increased resistance to fungal pathogens, e.g., of the familyPhakopsoraceae, for example soybean rust, is preferably a nucleic acidcoding for a CASAR protein, and is preferably as defined by SEQ ID NO:2, or a fragment, homolog, derivative, orthologue or paralogue thereof,or a splice variant thereof. Preferably, the nucleic acid coding for aCASAR protein of the present invention has at least 60% identity,preferably at least 70% sequence identity, at least 80%, at least 90%,at least 95%, at least 98%, at least 99% sequence identity, or even 100%sequence identity with SEQ ID NO: 2 or is a functional fragment thereof,or a splice variant thereof. Most preferred is at least 95% identity,more preferred is at least 98% or at least 99% identity with SEQ ID NO:2.

Preferably the CASAR nucleic acid is an isolated nucleic acid moleculeconsisting of or comprising a nucleic acid selected from the groupconsisting of:

-   (i) a nucleic acid having in increasing order of preference at least    60%, at least 61%, at least 62%, at least 63%, at least 64%, at    least 65%, at least 66%, at least 67%, at least 68%, at least 69%,    at least 70%, at least 71%, at least 72%, at least 73%, at least    74%, at least 75%, at least 76%, at least 77%, at least 78%, at    least 79%, at least 80%, at least 81%, at least 82%, at least 83%,    at least 84%, at least 85%, at least 86%, at least 87%, at least    88%, at least 89%, at least 90%, at least 91%, at least 92%, at    least 93%, at least 94%, at least 95%, at least 96%, at least 97%,    at least 98%, at least 99% or 100% sequence identity to the nucleic    acid sequence represented by SEQ ID NO: 2, 1, 5-12, 13, 15, 17, 19,    21, 23, 25, or 27, or a functional fragment, derivative, orthologue,    or paralogue thereof, or a splice variant thereof;-   (ii) a nucleic acid encoding a CASAR protein having in increasing    order of preference at least 60%, at least 61%, at least 62%, at    least 63%, at least 64%, at least 65%, at least 66%, at least 67%,    at least 68%, at least 69%, at least 70%, at least 71%, at least    72%, at least 73%, at least 74%, at least 75%, at least 76%, at    least 77%, at least 78%, at least 79%, at least 80%, at least 81%,    at least 82%, at least 83%, at least 84%, at least 85%, at least    86%, at least 87%, at least 88%, at least 89%, at least 90%, at    least 91%, at least 92%, at least 93%, at least 94%, at least 95%,    at least 96%, at least 97%, at least 98%, at least 99% or 100%    sequence identity to the amino acid sequence represented by SEQ ID    NO: 3, 14, 16, 18, 20, 22, 24, 26, or 28, or a functional fragment,    derivative, orthologue, or paralogue thereof; preferably the CASAR    protein has essentially the same biological activity as a CASAR    protein encoded by SEQ ID NO: 2 or 1; preferably the CASAR protein    confers enhanced fungal resistance relative to control plants;-   (iii) a nucleic acid molecule which hybridizes with a complementary    sequence of any of the nucleic acid molecules of (i) or (ii) under    high stringency hybridization conditions; preferably encoding a    CASAR protein; preferably wherein the nucleic acid molecule codes    for a polypeptide which has essentially identical properties to the    polypeptide described in SEQ ID NO: 3; preferably the encoded    protein confers enhanced fungal resistance relative to control    plants; and-   (iv) a nucleic acid encoding the same CASAR protein as the CASAR    nucleic acids of (i) to (iii) above, but differing from the CASAR    nucleic acids of (i) to (iii) above due to the degeneracy of the    genetic code.

Preferably, the nucleic acid coding for a CASAR protein of the presentinvention has at least 60% identity, preferably at least 70% sequenceidentity, at least 80%, at least 90%, at least 95%, at least 98%, atleast 99% sequence identity, or even 100% sequence identity with SEQ IDNO: 3, 14, 16, 18, 20, 22, 24, 26, or 28. Most preferred is at least 95%identity, more preferred is at least 98% or at least 99% identity withSEQ ID NO: 3, 14, 16, 18, 20, 22, 24, 26, or 28.

Preferably, the nucleic acid coding for a CASAR protein of the presentinvention has at least 60% identity, preferably at least 70% sequenceidentity, at least 80%, at least 90%, at least 95%, at least 98%, atleast 99% sequence identity, or even 100% sequence identity with SEQ IDNO: 3. Most preferred is at least 95% identity, more preferred is atleast 98% or at least 99% identity with SEQ ID NO: 3.

Preferably the CASAR nucleic acid is an isolated nucleic acid moleculeconsisting of or comprising a nucleic acid selected from the groupconsisting of:

-   (i) a nucleic acid having in increasing order of preference at least    60%, at least 61%, at least 62%, at least 63%, at least 64%, at    least 65%, at least 66%, at least 67%, at least 68%, at least 69%,    at least 70%, at least 71%, at least 72%, at least 73%, at least    74%, at least 75%, at least 76%, at least 77%, at least 78%, at    least 79%, at least 80%, at least 81%, at least 82%, at least 83%,    at least 84%, at least 85%, at least 86%, at least 87%, at least    88%, at least 89%, at least 90%, at least 91%, at least 92%, at    least 93%, at least 94%, at least 95%, at least 96%, at least 97%,    at least 98%, at least 99% or 100% sequence identity to the nucleic    acid sequence represented by SEQ ID NO: 2, or a functional fragment,    derivative, orthologue, or paralogue thereof, or a splice variant    thereof;-   (ii) a nucleic acid encoding a CASAR protein having in increasing    order of preference at least 60%, at least 61%, at least 62%, at    least 63%, at least 64%, at least 65%, at least 66%, at least 67%,    at least 68%, at least 69%, at least 70%, at least 71%, at least    72%, at least 73%, at least 74%, at least 75%, at least 76%, at    least 77%, at least 78%, at least 79%, at least 80%, at least 81%,    at least 82%, at least 83%, at least 84%, at least 85%, at least    86%, at least 87%, at least 88%, at least 89%, at least 90%, at    least 91%, at least 92%, at least 93%, at least 94%, at least 95%,    at least 96%, at least 97%, at least 98%, at least 99% or 100%    sequence identity to the amino acid sequence represented by SEQ ID    NO: 3, or a functional fragment, derivative, orthologue, or    paralogue thereof; preferably the CASAR protein has essentially the    same biological activity as a CASAR protein encoded by SEQ ID NO: 2,    preferably the CASAR protein confers enhanced fungal resistance    relative to control plants;-   (iii) a nucleic acid molecule which hybridizes with a complementary    sequence of any of the nucleic acid molecules of (i) or (ii) under    high stringency hybridization conditions; preferably encoding a    CASAR protein; preferably wherein the nucleic acid molecule codes    for a polypeptide which has essentially identical properties to the    polypeptide described in SEQ ID NO: 3; preferably the encoded    protein confers enhanced fungal resistance relative to control    plants; and-   (iv) a nucleic acid encoding the same CASAR protein as the CASAR    nucleic acids of (i) to (iii) above, but differing from the CASAR    nucleic acids of (i) to (iii) above due to the degeneracy of the    genetic code.

Percentages of identity of a nucleic acid are indicated with referenceto the entire nucleotide region given in a sequence specificallydisclosed herein.

Preferably the portion of the CASAR nucleic acid is about 150-160, about160-170, about 170-180, about 180-190, about 190-200, about 200-210,about 210-220, about 220-230, about 230-240, about 240-250, or about250-261 nucleotides, preferably consecutive nucleotides, preferablycounted from the 5′ or 3′ end of the nucleic acid, in length, of thenucleic acid sequences given in SEQ ID NO: 2, 1, 5-12, 13, 15, 17, 19,21, 23, 25, or 27.

Preferably, the CASAR nucleic acid comprises at least about 50, at leastabout 75, at least about 100, at least about 125, at least about 150, atleast about 160, at least about 170, at least about 180, at least about190, at least about 200, at least about 210, at least about 220, atleast about 230, at least about 240, or at least about 250 nucleotides,preferably continuous nucleotides, preferably counted from the 5′ or 3′end of the nucleic acid or up to the full length of the nucleic acidsequence set out in SEQ ID NO: 2, 1, 5-12, 13, 15, 17, 19, 21, 23, 25,or 27.

Preferably the portion of the CASAR nucleic acid is about 150-160, about160-170, about 170-180, about 180-190, about 190-200, about 200-210,about 210-220, about 220-230, about 230-240, about 240-250, or about250-261 nucleotides, preferably consecutive nucleotides, preferablycounted from the 5′ or 3′ end of the nucleic acid, in length, of thenucleic acid sequences given in SEQ ID NO: 2 or 1.

Preferably, the CASAR nucleic acid comprises at least about 50, at leastabout 75, at least about 100, at least about 125, at least about 150, atleast about 160, at least about 170, at least about 180, at least about190, at least about 200, at least about 210, at least about 220, atleast about 230, at least about 240, or at least about 250 nucleotides,preferably continuous nucleotides, preferably counted from the 5′ or 3′end of the nucleic acid or up to the full length of the nucleic acidsequence set out in SEQ ID NO: 2 or 1.

All the nucleic acid sequences mentioned herein (single-stranded anddouble-stranded DNA and RNA sequences, for example cDNA and mRNA) can beproduced in a known way by chemical synthesis from the nucleotidebuilding blocks, e.g. by fragment condensation of individualoverlapping, complementary nucleic acid building blocks of the doublehelix. Chemical synthesis of oligonucleotides can, for example, beperformed in a known way, by the phosphoamidite method (Voet, Voet, 2ndedition, Wiley Press, New York, pages 896-897). The accumulation ofsynthetic oligonucleotides and filling of gaps by means of the Klenowfragment of DNA polymerase and ligation reactions as well as generalcloning techniques are described in Sambrook et al. (1989), see below.

The CASAR nucleic acids described herein are useful in the constructs,methods, plants, harvestable parts and products of the invention.

CASAR Proteins

The CASAR protein is preferably defined by SEQ ID NO: 3, 14, 16, 18, 20,22, 24, 26, or 28, or a fragment, homolog, derivative, orthologue orparalogue thereof. Preferably, the CASAR protein of the presentinvention is encoded by a nucleic acid, which has at least 60% identity,preferably at least 70% sequence identity, at least 80%, at least 90%,at least 95%, at least 98%, at least 99% sequence identity, or even 100%sequence identity with SEQ ID NO: 3, 14, 16, 18, 20, 22, 24, 26, or 28or a functional fragment thereof. More preferably, the CASAR protein ofthe present invention has at least 60%, preferably at least 70% sequenceidentity, at least 80%, at least 90%, at least 95%, at least 98%, atleast 99% sequence identity, or even 100% sequence identity with SEQ IDNO: 3, 14, 16, 18, 20, 22, 24, 26, or 28, or is a functional fragmentthereof, an orthologue or a paralogue thereof. Most preferred is atleast 90% identity, least 95% identity, more preferred is at least 98%or at least 99% identity with SEQ ID NO: 3, 14, 16, 18, 20, 22, 24, 26,or 28.

The CASAR protein is preferably defined by SEQ ID NO: 3, or a fragment,homolog, derivative, orthologue or paralogue thereof. Preferably, theCASAR protein of the present invention is encoded by a nucleic acid,which has at least 60% identity, preferably at least 70% sequenceidentity, at least 80%, at least 90%, at least 95%, at least 98%, atleast 99% sequence identity, or even 100% sequence identity with SEQ IDNO: 3, or a functional fragment thereof. More preferably, the CASARprotein of the present invention has at least 60%, preferably at least70% sequence identity, at least 80%, at least 90%, at least 95%, atleast 98%, at least 99% sequence identity, or even 100% sequenceidentity with SEQ ID NO: 3, or is a functional fragment thereof, anorthologue or a paralogue thereof. Most preferred is at least 90%identity, least 95% identity, more preferred is at least 98% or at least99% identity with SEQ ID NO: 3.

Preferably, the CASAR protein is a protein consisting of or comprisingan amino acid sequence selected from the group consisting of:

-   (i) an amino acid sequence having in increasing order of preference    at least 60%, at least 61%, at least 62%, at least 63%, at least    64%, at least 65%, at least 66%, at least 67%, at least 68%, at    least 69%, at least 70%, at least 71%, at least 72%, at least 73%,    at least 74%, at least 75%, at least 76%, at least 77%, at least    78%, at least 79%, at least 80%, at least 81%, at least 82%, at    least 83%, at least 84%, at least 85%, at least 86%, at least 87%,    at least 88%, at least 89%, at least 90%, at least 91%, at least    92%, at least 93%, at least 94%, at least 95%, at least 96%, at    least 97%, at least 98%, at least 99% or 100% sequence identity to    the amino acid sequence represented by SEQ ID NO: 3, 14, 16, 18, 20,    22, 24, 26, or 28, or a functional fragment, derivative, orthologue,    or paralogue thereof; preferably the CASAR protein has essentially    the same biological activity as a CASAR protein encoded by SEQ ID    NO: 2 or 1; preferably the CASAR protein confers enhanced fungal    resistance relative to control plants; or-   (ii) an amino acid sequence encoded by a nucleic acid having in    increasing order of preference at least 60%, at least 61%, at least    62%, at least 63%, at least 64%, at least 65%, at least 66%, at    least 67%, at least 68%, at least 69%, at least 70%, at least 71%,    at least 72%, at least 73%, at least 74%, at least 75%, at least    76%, at least 77%, at least 78%, at least 79%, at least 80%, at    least 81%, at least 82%, at least 83%, at least 84%, at least 85%,    at least 86%, at least 87%, at least 88%, at least 89%, at least    90%, at least 91%, at least 92%, at least 93%, at least 94%, at    least 95%, at least 96%, at least 97%, at least 98%, at least 99% or    100% sequence identity to the nucleic acid sequence represented by    SEQ ID NO: 2, 1, 5-12, 13, 15, 17, 19, 21, 23, 25, or 27, or a    functional fragment, derivative, orthologue, or paralogue thereof,    or a splice variant thereof; preferably the CASAR protein confers    enhanced fungal resistance relative to control plants.

Preferably, the CASAR protein is a protein comprising an amino acidsequence selected from the group consisting of:

-   (i) an amino acid sequence having in increasing order of preference    at least 80%, at least 81%, at least 82%, at least 83%, at least    84%, at least 85%, at least 86%, at least 87%, at least 88%, at    least 89%, at least 90%, at least 91%, at least 92%, at least 93%,    at least 94%, at least 95%, at least 96%, at least 97%, at least    98%, at least 99% or 100% sequence identity to the amino acid    sequence represented by SEQ ID NO: 3, or a functional fragment,    derivative, orthologue, or paralogue thereof; preferably the CASAR    protein has essentially the same biological activity as a CASAR    protein encoded by SEQ ID NO: 2 or 1; preferably the CASAR protein    confers enhanced fungal resistance relative to control plants; or-   (ii) an amino acid sequence encoded by a nucleic acid having in    increasing order of preference at least 80%, at least 81%, at least    82%, at least 83%, at least 84%, at least 85%, at least 86%, at    least 87%, at least 88%, at least 89%, at least 90%, at least 91%,    at least 92%, at least 93%, at least 94%, at least 95%, at least    96%, at least 97%, at least 98%, at least 99% or 100% sequence    identity to the nucleic acid sequence represented by SEQ ID NO: 2 or    1, or a functional fragment, derivative, orthologue, or paralogue    thereof, or a splice variant thereof; preferably the CASAR protein    confers enhanced fungal resistance relative to control plants.

A preferred derivative of a CASAR protein is a CASAR protein consistingof or comprising an amino acid sequence selected from the groupconsisting of:

an amino acid sequence having in increasing order of preference at least60%, at least 61%, at least 62%, at least 63%, at least 64%, at least65%, at least 66%, at least 67%, at least 68%, at least 69%, at least70%, at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, and at least 99% sequenceidentity to the amino acid sequence represented by SEQ ID NO: 3, 14, 16,18, 20, 22, 24, 26, or 28,wherein the non-identical amino acid residues are conservative aminoacid substitutions, preferably as shown in Table 1, of the correspondingamino acid residue of SEQ ID NO: 3, 14, 16, 18, 20, 22, 24, 26, or 28;preferably the CASAR protein has essentially the same biologicalactivity as SEQ ID NO: 3, 14, 16, 18, 20, 22, 24, 26, or 28 or as aCASAR protein encoded by SEQ ID NO: 2 or 1; preferably the CASAR proteinconfers enhanced fungal resistance relative to control plants.

Preferably, the CASAR protein consists of or comprises an amino acidsequence represented by SEQ ID NO: 3, 14, 16, 18, 20, 22, 24, 26, or 28with one or more conservative amino acid substitutions, preferably asshown in Table 1, of the corresponding amino acid residues of SEQ ID NO:3, 14, 16, 18, 20, 22, 24, 26, or 28. Preferably 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 1-10, 10-20, 20-30, 40-50, 50-60, 60-70, 70-80, 80-90,90-100, 100-110, 110-120, or 120-130 amino acid residues of SEQ ID NO: 3are conservative amino acid substitutions, preferably as shown in Table1, of the corresponding amino acid residue of SEQ ID NO: 3, 14, 16, 18,20, 22, 24, 26, or 28.

More preferably, the CASAR protein consists of or comprises an aminoacid sequence having at least 80%, at least 85%, at least 90%, at least95%, at least 98% or at least 99% sequence identity with an amino acidsequence as represented by SEQ ID NO: 3, 14, 16, 18, 20, 22, 24, 26, or28, wherein at least 1, at least 2, at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, at least22, at least 23, at least 24, at least 25, at least 26, at least 27, atleast 28, at least 29, or at least 30 of the non-identical amino acidresidues, or wherein 1-10, 10-20, 20-30, 40-50, 50-60, 60-70, 70-80,80-90 or 90-100 or even all of the non-identical amino acid residues areconservative amino acid substitutions, preferably as shown in Table 1,of the corresponding amino acid residue of SEQ ID NO: 3, 14, 16, 18, 20,22, 24, 26, or 28.

Percentages of identity of a polypeptide or protein are indicated withreference to the entire amino acid sequence specifically disclosedherein.

Preferably, the CASAR protein comprises at least about 25, at leastabout 30, at least about 35, at least about 40, at least about 45, atleast about 50, at least about 55, at least about 60, at least about 65,at least about 70, at least about 75, at least about 80, or at leastabout 85 preferably continuous amino acid residues, preferably countedfrom the N-terminus or the C-terminus of the amino acid sequence, or upto the full length of the amino acid sequence set out in SEQ ID NO: 3,14, 16, 18, 20, 22, 24, 26, or 28.

Preferably, the CASAR polypeptide comprises about 25-30, about 30-35,about 35-40, about 40-45, about 45-50, about 50-55, about 55-60, about60-65, about 65-70, about 70-75, about 75-80, or about 80-86 aminoacids, preferably consecutive amino acids, preferably counted from theN-terminus or C-terminus of the amino acid sequence, or up to the fulllength of any of the amino acid sequences encoded by the nucleic acidsequences set out in SEQ ID NO: 3, 14, 16, 18, 20, 22, 24, 26, or 28.

Preferably, the CASAR protein comprises at least about 25, at leastabout 30, at least about 35, at least about 40, at least about 45, atleast about 50, at least about 55, at least about 60, at least about 65,at least about 70, at least about 75, at least about 80, or at leastabout 85 preferably continuous amino acid residues, preferably countedfrom the N-terminus or the C-terminus of the amino acid sequence, or upto the full length of the amino acid sequence set out in SEQ ID NO: 3.

Preferably, the CASAR polypeptide comprises about 25-30, about 30-35,about 35-40, about 40-45, about 45-50, about 50-55, about 55-60, about60-65, about 65-70, about 70-75, about 75-80, or about 80-86 aminoacids, preferably consecutive amino acids, preferably counted from theN-terminus or C-terminus of the amino acid sequence, or up to the fulllength of any of the amino acid sequences encoded by the nucleic acidsequences set out in SEQ ID NO: 3.

The CASAR proteins described herein are useful in the constructs,methods, plants, harvestable parts and products of the invention.

Methods for Increasing Fungal Resistance; Methods for Modulating GeneExpression

One embodiment of the invention is a method for increasing fungalresistance, preferably resistance to Phakopsoraceae, for example soybean rust, in a plant, plant part, or plant cell by increasing theexpression of a CASAR protein or a functional fragment, orthologue,paralogue or homologue thereof in comparison to wild-type plants,wild-type plant parts or wild-type plant cells.

The present invention also provides a method for increasing resistanceto fungal pathogens, preferably to a heminecrotrophic pathogen, inparticular to rust pathogens (i.e., fungal pathogens of the orderPucciniales), preferably fungal pathogens of the family Phakopsoraceae,preferably against fungal pathogens of the genus Phakopsora, mostpreferably against Phakopsora pachyrhizi and Phakopsora meibomiae, alsoknown as soy bean rust in plants or plant cells, wherein in comparisonto wild type plants, wild type plant parts, or wild type plant cells aCASAR protein is overexpressed.

The present invention further provides a method for increasingresistance to fungal pathogens of the genus Phakopsora, most preferablyagainst Phakopsora pachyrhizi and Phakopsora meibomiae, also known assoy bean rust in plants or plant cells by overexpression of a CASARprotein.

In preferred embodiments, the protein amount and/or function of theCASAR protein in the plant is increased by at least 10%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 95% or more in comparison to a wildtype plant that is not transformed with the CASAR nucleic acid.

In one embodiment of the invention, the CASAR protein is encoded by

-   (i) an exogenous nucleic acid having at least 60%, preferably at    least 70%, for example at least 75%, more preferably at least 80%,    for example at least 85%, even more preferably at least 90%, for    example at least 95% or at least 96% or at least 97% or at least    98%, most preferably 99% identity with SEQ ID NO: 2, 1, 5-12, 13,    15, 17, 19, 21, 23, 25, or 27, a functional fragment thereof, or an    orthologue or a paralogue thereof, or a splice variant thereof; or    by-   (ii) an exogenous nucleic acid encoding a protein having at least    60% identity, preferably at least 70%, for example at least 75%,    more preferably at least 80%, for example at least 85%, even more    preferably at least 90%, for example at least 95% or at least 96% or    at least 97% or at least 98% most preferably 99% homology with SEQ    ID NO: 3, 14, 16, 18, 20, 22, 24, 26, or 28, a functional fragment    thereof, an orthologue or a paralogue thereof, preferably the    encoded protein confers enhanced fungal resistance relative to    control plants;-   (iii) an exogenous nucleic acid capable of hybridizing under    stringent conditions with a complementary sequence of any of the    nucleic acids according to (i) or (ii); preferably encoding a CASAR    protein; preferably wherein the nucleic acid molecule codes for a    polypeptide which has essentially identical properties to the    polypeptide described in SEQ ID NO: 3; preferably the encoded    protein confers enhanced fungal resistance relative to control    plants; or by-   (iv) an exogenous nucleic acid encoding the same CASAR protein as    any of the nucleic acids of (i) to (iii) above, but differing from    the nucleic acids of (i) to (iii) above due to the degeneracy of the    genetic code.

A method for increasing fungal resistance, preferably resistance toPhakopsoraceae, for example soy bean rust, in a plant, plant part, orplant cell, by increasing the expression of a CASAR protein or afunctional fragment, orthologue, paralogue or homologue thereof, or asplice variant thereof, wherein the CASAR protein is encoded by

-   (i) an exogenous nucleic acid having at least 60% identity,    preferably at least 70% sequence identity, at least 80%, at least    90%, at least 95%, at least 98%, at least 99% sequence identity, or    even 100% sequence identity with SEQ ID NO: 2, 1, 5-12, 13, 15, 17,    19, 21, 23, 25, or 27, or a functional fragment thereof, an    orthologue or a paralogue thereof, or a splice variant thereof;-   (ii) an exogenous nucleic acid encoding a protein having at least    60%, preferably at least 70% sequence identity, at least 80%, at    least 90%, at least 95%, at least 98%, at least 99% sequence    identity, or even 100% sequence identity with SEQ ID NO: 3, 14, 16,    18, 20, 22, 24, 26, or 28, a functional fragment thereof, an    orthologue or a paralogue thereof; preferably the encoded protein    confers enhanced fungal resistance relative to control plants;-   (iii) an exogenous nucleic acid capable of hybridizing under    stringent conditions with a complementary sequence of any of the    nucleic acids according to (i) or (ii); preferably encoding a CASAR    protein; preferably wherein the nucleic acid molecule codes for a    polypeptide which has essentially identical properties to the    polypeptide described in SEQ ID NO: 3; preferably the encoded    protein confers enhanced fungal resistance relative to control    plants; and/or by-   (iv) an exogenous nucleic acid encoding the same CASAR protein as    any of the nucleic acids of (i) to (iii) above, but differing from    the nucleic acids of (i) to (iii) above due to the degeneracy of the    genetic code    is a further embodiment of the invention.

A method for increasing fungal resistance, preferably resistance toPhakopsoraceae, for example soy bean rust, in a plant, plant part, orplant cell, by increasing the expression of a CASAR protein or afunctional fragment, orthologue, paralogue or homologue thereof, or asplice variant thereof, wherein the CASAR protein is encoded by

-   (i) an exogenous nucleic acid having at least 60% identity,    preferably at least 70% sequence identity, at least 80%, at least    90%, at least 95%, at least 98%, at least 99% sequence identity, or    even 100% sequence identity with SEQ ID NO: 2 or a functional    fragment thereof, an orthologue or a paralogue thereof, or a splice    variant thereof;-   (ii) an exogenous nucleic acid encoding a protein having at least    60%, preferably at least 70% sequence identity, at least 80%, at    least 90%, at least 95%, at least 98%, at least 99% sequence    identity, or even 100% sequence identity with SEQ ID NO: 3, a    functional fragment thereof, an orthologue or a paralogue thereof;    preferably the encoded protein confers enhanced fungal resistance    relative to control plants;-   (iii) an exogenous nucleic acid capable of hybridizing under    stringent conditions with a complementary sequence of any of the    nucleic acids according to (i) or (ii); preferably encoding a CASAR    protein; preferably wherein the nucleic acid molecule codes for a    polypeptide which has essentially identical properties to the    polypeptide described in SEQ ID NO: 3; preferably the encoded    protein confers enhanced fungal resistance relative to control    plants; and/or by-   (iv) an exogenous nucleic acid encoding the same CASAR protein as    any of the nucleic acids of (i) to (iii) above, but differing from    the nucleic acids of (i) to (iii) above due to the degeneracy of the    genetic code    is a further embodiment of the invention.

In a further method of the invention, the method comprises the steps of

-   (a) stably transforming a plant cell with a recombinant expression    cassette comprising    -   (i) a nucleic acid having at least 60% identity, preferably at        least 70% sequence identity, at least 80%, at least 90%, at        least 95%, at least 98%, at least 99% sequence identity, or even        100% sequence identity with SEQ ID NO: 2, 1, 5-12, 13, 15, 17,        19, 21, 23, 25, or 27, or a functional fragment thereof, or an        orthologue or a paralogue thereof, or a splice variant thereof;    -   (ii) a nucleic acid coding for a protein having at least 60%        identity, preferably at least 70% sequence identity, at least        80%, at least 90%, at least 95%, at least 98%, at least 99%        sequence identity, or even 100% sequence identity with SEQ ID        NO: 3, 14, 16, 18, 20, 22, 24, 26, or 28, a functional fragment        thereof, an orthologue or a paralogue thereof; preferably the        encoded protein confers enhanced fungal resistance relative to        control plants;    -   (iii) a nucleic acid capable of hybridizing under stringent        conditions with a complementary sequence of any of the nucleic        acids according to (i) or (ii); preferably encoding a CASAR        protein; preferably wherein the nucleic acid molecule codes for        a polypeptide which has essentially identical properties to the        polypeptide described in SEQ ID NO: 3; preferably the encoded        protein confers enhanced fungal resistance relative to control        plants; and/or    -   (iv) a nucleic acid encoding the same CASAR polypeptide as any        of the nucleic acids of (i) to (iii) above, but differing from        the nucleic acids of (i) to (iii) above due to the degeneracy of        the genetic code,    -   in functional linkage with a promoter;-   (b) regenerating the plant from the plant cell; and-   (c) expressing said nucleic acid, optionally wherein the nucleic    acid which codes for a CASAR protein is expressed in an amount and    for a period sufficient to generate or to increase soybean rust    resistance in said plant.

Preferably, the method comprises the steps of

-   (a) stably transforming a plant cell with a recombinant expression    cassette comprising    -   (i) a nucleic acid having at least 60% identity, preferably at        least 70% sequence identity, at least 80%, at least 90%, at        least 95%, at least 98%, at least 99% sequence identity, or even        100% sequence identity with SEQ ID NO: 2, or a functional        fragment thereof, or an orthologue or a paralogue thereof, or a        splice variant thereof;    -   (ii) a nucleic acid coding for a protein having at least 60%        identity, preferably at least 70% sequence identity, at least        80%, at least 90%, at least 95%, at least 98%, at least 99%        sequence identity, or even 100% sequence identity with SEQ ID        NO: 3, a functional fragment thereof, an orthologue or a        paralogue thereof; preferably the encoded protein confers        enhanced fungal resistance relative to control plants;    -   (iii) a nucleic acid capable of hybridizing under stringent        conditions with a complementary sequence of any of the nucleic        acids according to (i) or (ii); preferably encoding a CASAR        protein; preferably wherein the nucleic acid molecule codes for        a polypeptide which has essentially identical properties to the        polypeptide described in SEQ ID NO: 3; preferably the encoded        protein confers enhanced fungal resistance relative to control        plants; and/or    -   (iv) a nucleic acid encoding the same CASAR polypeptide as any        of the nucleic acids of (i) to (iii) above, but differing from        the nucleic acids of (i) to (iii) above due to the degeneracy of        the genetic code,    -   in functional linkage with a promoter;-   (b) regenerating the plant from the plant cell; and-   (c) expressing said nucleic acid, optionally wherein the nucleic    acid which codes for a CASAR protein is expressed in an amount and    for a period sufficient to generate or to increase soybean rust    resistance in said plant.

Preferably, the promoter is a rust induced and/or mesophyll-specificpromoter, preferably the rust induced mesophyll specific promoter 820,preferably, as shown in SEQ ID NO: 4.

Preferably, the method for increasing fungal resistance, preferablyresistance to Phakopsoraceae, for example soy bean rust, in a plant,plant part, or plant cell further comprises the step of selecting atransgenic plant expressing

-   (i) an exogenous nucleic acid having at least 60% identity,    preferably at least 70% sequence identity, at least 80%, at least    90%, at least 95%, at least 98%, at least 99% sequence identity, or    even 100% sequence identity with SEQ ID NO: 2, 1, 5-12, 13, 15, 17,    19, 21, 23, 25, or 27, or a functional fragment thereof, or an    orthologue or a paralogue thereof, or a splice variant thereof;-   (ii) an exogenous nucleic acid coding for a protein having at least    60% identity, preferably at least 70% sequence identity, at least    80%, at least 90%, at least 95%, at least 98%, at least 99% sequence    identity, or even 100% sequence identity with SEQ ID NO: 3, 14, 16,    18, 20, 22, 24, 26, or 28, a functional fragment thereof, an    orthologue or a paralogue thereof; preferably the encoded protein    confers enhanced fungal resistance relative to control plants;-   (iii) an exogenous nucleic acid capable of hybridizing under    stringent conditions a complementary sequence of any of the nucleic    acids according to (i) or (ii); preferably encoding a CASAR protein;    preferably wherein the nucleic acid molecule codes for a polypeptide    which has essentially identical properties to the polypeptide    described in SEQ ID NO: 3; preferably the encoded protein confers    enhanced fungal resistance relative to control plants; and/or-   (iv) an exogenous nucleic acid encoding the same CASAR polypeptide    as any of the nucleic acids of (i) to (iii) above, but differing    from the nucleic acids of (i) to (iii) above due to the degeneracy    of the genetic code.

A preferred embodiment is a method for increasing resistance to soy beanrust in a soy bean plant, soy bean plant part, or soy bean plant cell,by increasing the expression of a CASAR protein, wherein the CASARprotein is encoded by

-   (i) an exogenous nucleic acid having at least 80%, at least 90%, at    least 95%, at least 98%, at least 99% sequence identity, or even    100% sequence identity with SEQ ID NO: 2, 1, 5-12, 13, 15, 17, 19,    21, 23, 25, or 27;-   (ii) an exogenous nucleic acid encoding a protein having at least    80%, at least 90%, at least 95%, at least 98%, at least 99% sequence    identity, or even 100% sequence identity with SEQ ID NO: 3, 14, 16,    18, 20, 22, 24, 26, or 28; preferably the encoded protein confers    enhanced fungal resistance relative to control plants;-   (iii) an exogenous nucleic acid capable of hybridizing under    stringent conditions with a complementary sequence of any of the    nucleic acids according to (i) or (ii); preferably encoding a CASAR    protein; preferably wherein the nucleic acid molecule codes for a    polypeptide which has essentially identical properties to the    polypeptide described in SEQ ID NO: 3; preferably the encoded    protein confers enhanced fungal resistance relative to control    plants; and/or by-   (iv) an exogenous nucleic acid encoding the same CASAR protein as    any of the nucleic acids of (i) to (iii) above, but differing from    the nucleic acids of (i) to (iii) above due to the degeneracy of the    genetic code,    wherein increasing the expression of the CASAR protein is achieved    by transforming the soy bean plant, plant part or plant cell with a    nucleic acid comprising the nucleic acid set out under item (i)    or (ii) or (iii) or (iv).

Also a preferred embodiment is a method for increasing resistance to soybean rust in a soy bean plant, soy bean plant part, or soy bean plantcell, by increasing the expression of a CASAR protein, wherein the CASARprotein is encoded by

-   (i) an exogenous nucleic acid having at least 80%, at least 90%, at    least 95%, at least 98%, at least 99% sequence identity, or even    100% sequence identity with SEQ ID NO: 2, 1, 5-12, 13, 15, 17, 19,    21, 23, 25, or 27;-   (ii) an exogenous nucleic acid encoding a protein having at least    80%, at least 90%, at least 95%, at least 98%, at least 99% sequence    identity, or even 100% sequence identity with SEQ ID NO: 3, 14, 16,    18, 20, 22, 24, 26, or 28; preferably the encoded protein confers    enhanced fungal resistance relative to control plants; or-   (iii) an exogenous nucleic acid encoding the same CASAR protein as    any of the nucleic acids of (i) to (ii) above, but differing from    the nucleic acids of (i) to (ii) above due to the degeneracy of the    genetic code,    wherein increasing the expression of the CASAR protein is achieved    by transforming the soy bean plant, plant part or plant cell with a    nucleic acid comprising the nucleic acid set out under item (i)    or (ii) or (iii).

The fungal pathogens or fungus-like pathogens (such as, for example,Chromista) can belong to the group comprising Plasmodiophoramycota,Oomycota, Ascomycota, Chytridiomycetes, Zygomycetes, Basidiomycota orDeuteromycetes (Fungi imperfecti). Pathogens which may be mentioned byway of example, but not by limitation, are those detailed in Tables 2and 3, and the diseases which are associated with them.

TABLE 2 Diseases caused by biotrophic and/or heminecrotrophicphytopathogenic fungi Disease Pathogen Leaf rust Puccinia reconditaYellow rust P. striiformis Powdery mildew Erysiphe graminis/Blumeriagrarrinis Rust (common corn) Puccinia sorghi Rust (Southern corn)Puccinia polysora Tobacco leaf spot Cercospora nicotianae Rust (soybean)Phakopsora pachyrhizi, P. meibomia

Rust (tropical corn) Physopella pallescens, P. zeae = Angiopsora zea

indicates data missing or illegible when filed

TABLE 3 Diseases caused by necrotrophic and/or hemibiotrophic fungi andOomycetes Disease Pathogen Plume blotch Septoria (Stagonospora) nodorurrLeaf blotch Septoria tritici Ear fusarioses Fusarium spp. Late blightPhytophthora infestans Anthrocnose leaf blight Colletotrichumgraminicola (teleomorph: Glomerella Anthracnose stalk rot graminicolaPolitis); Glomerella tucumanensis (anamorph: Glomerella falcatum Went)Curvularia leaf spot Curvularia clavata, C. eragrostidis, = C. maculans(teleomorph: Cochliobolus eragrostidis), Curvularia inaequalis, C.intermedia (teleomorph: Cochliobolus intermedius), Curvularia lunata(teleomorph: Cochliobolus lunatus), Curvularia pallescens (teleomorph:Cochliobolus pallescens), Curvularia senegalensis, C. tuberculata(teleomorph: Cochliobolus tuberculatus) Didymella leaf spot Didymellaexitalis Diplodia leaf spot or streak Stenocarpella macrospora =Diplodialeaf macrospora Brown stripe downy mildew Sclerophthora rayssiaevar. zea

Crazy top downy mildew Sclerophthora macrospora = Sclerospora macrosporaGreen ear downy mildew Sclerospora graminicola (graminicola downymildew) Leaf spots, minor Alternaria alternata, Ascochyta maydis, A.tritici, A. zeicola, Bipolaris victoriae = Helminthosporium victoriae(teleomorph: Cochliobolus victoriae), C. sativus (anamorph: Bipolarissorokiniana = H. sorokinianum = H. sativum), Epicoccum nigrum,Exserohilum prolatum = Drechslera prolata (teleomorph: Setosphaeriaprolata) Graphium penicillioides, Leptosphaeria maydis, Leptothyriumzeae, Ophiosphaerella herpotricha, (anamorph: Scolecosporiella sp.),Paraphaeosphaeria michotii, Phoma sp., Septoria zeae, S. zeicola, S.zeina Northern corn leaf blight (white Setosphaeria turcica (anamorph:Exserohilum turcicum = blast, crown stalk rot, stripe) Helminthosporiumturcicum) Northern corn leaf spot Cochliobolus carbonum (anamorph:Bipolaris zeicola = Helminthosporium ear rot (race 1) Helminthosporiumcarbonum) Phaeosphaeria leaf spot Phaeosphaeria maydis = Sphaerulinamaydi

Rostratum leaf spot Setosphaeria rostrata, (anamorph: xserohilumrostratum = (Helminthosporium leaf Helminthosporium rostratum) disease,ear and stalk rot) Java downy mildew Peronosclerospora maydis =Sclerospora maydis Philippine downy mildew Peronosclerosporaphilippinensis = Sclerospora philippinensis Sorghum downy mildewPeronosclerospora sorghi = Sclerospora sorghi Spontaneum downy mildewPeronosclerospora spontanea = Sclerospora spontanea Sugarcane downymildew Peronosclerospora sacchari = Sclerospora sacchari Sclerotium earrot (southern blight) Sclerotium rolfsii Sacc. (teleomorph: Atheliarolfsii) Seed rot-seedling blight Bipolaris sorokiniana, B. zeicola =Helminthosporium carbonum, Diplodia maydis, Exserohilum pedicillatum,Exserohilum turcicum = Helminthosporium turcicum, Fusarium avenaceum, F.culmorum, F. moniliforme, Gibberella zeae (anamorph: F. graminearum),Macrophomina phaseolina, Penicillium spp., Phomopsis sp., Pythium spp.,Rhizoctonia solani, R. zeae, Sclerotium rolfsii, Spicaria sp.Selenophoma leaf spot Selenophoma sp. Yellow leaf blight Ascochytaischaemi, Phyllosticta maydis (teleomorph: Mycosphaerella zeae-maydis)Zonate leaf spot Gloeocercospora sorghi

indicates data missing or illegible when filed

The following are especially preferred:

-   -   Plasmodiophoromycota such as Plasmodiophora brassicae (clubroot        of crucifers), Spongospora subterranea, Polymyxa graminis,    -   Oomycota such as Bremia lactucae (downy mildew of lettuce),        Peronospora (downy mildew) in snapdragon (P. antirrhini), onion        (P. destructor), spinach (P. effusa), soybean (P. manchurica),        tobacco (“blue mold”; P. tabacina) alfalfa and clover (P.        trifolium), Pseudoperonospora humuli (downy mildew of hops),        Plasmopara (downy mildew in grapevines) (P. viticola) and        sunflower (P. halstedii), Sclerophthora macrospora (downy mildew        in cereals and grasses), Pythium (for example damping-off of        Beta beet caused by P. debaryanum), Phytophthora infestans (late        blight in potato and in tomato and the like), Albugo spec.    -   Ascomycota such as Microdochium nivale (snow mold of rye and        wheat), Fusarium, Fusarium graminearum, Fusarium culmorum        (partial ear sterility mainly in wheat), Fusarium oxysporum        (Fusarium wilt of tomato), Blumeria graminis (powdery mildew of        barley (f.sp. hordei) and wheat (f.sp. tritici)), Erysiphe pisi        (powdery mildew of pea), Nectria galligena (Nectria canker of        fruit trees), Uncinula necator (powdery mildew of grapevine),        Pseudopeziza tracheiphila (red fire disease of grapevine),        Claviceps purpurea (ergot on, for example, rye and grasses),        Gaeumannomyces graminis (take-all on wheat, rye and other        grasses), Magnaporthe grisea, Pyrenophora graminea (leaf stripe        of barley), Pyrenophora teres (net blotch of barley),        Pyrenophora tritici-repentis (leaf blight of wheat), Venturia        inaequalis (apple scab), Sclerotinia sclerotium (stalk break,        stem rot), Pseudopeziza medicaginis (leaf spot of alfalfa, white        and red clover).    -   Basidiomycetes such as Typhula incarnata (typhula blight on        barley, rye, wheat), Ustilago maydis (blister smut on maize),        Ustilago nuda (loose smut on barley), Ustilago tritici (loose        smut on wheat, spelt), Ustilago avenae (loose smut on oats),        Rhizoctonia solani (rhizoctonia root rot of potato),        Sphacelotheca spp. (head smut of sorghum), Melampsora lini (rust        of flax), Puccinia graminis (stem rust of wheat, barley, rye,        oats), Puccinia recondita (leaf rust on wheat), Puccinia        dispersa (brown rust on rye), Puccinia hordei (leaf rust of        barley), Puccinia coronata (crown rust of oats), Puccinia        striiformis (yellow rust of wheat, barley, rye and a large        number of grasses), Uromyces appendiculatus (brown rust of        bean), Sclerotium rolfsii (root and stem rots of many plants).    -   Deuteromycetes (Fungi imperfecti) such as Septoria        (Stagonospora) nodorum (glume blotch) of wheat (Septoria        tritici), Pseudocercosporella herpotrichoides (eyespot of wheat,        barley, rye), Rynchosporium secalis (leaf spot on rye and        barley), Alternaria solani (early blight of potato, tomato),        Phoma betae (blackleg on Beta beet), Cercospora beticola (leaf        spot on Beta beet), Alternaria brassicae (black spot on oilseed        rape, cabbage and other crucifers), Verticillium dahliae        (verticillium wilt), Colletotrichum, Colletotrichum        lindemuthianum (bean anthracnose), Phoma lingam (blackleg of        cabbage and oilseed rape), Botrytis cinerea (grey mold of        grapevine, strawberry, tomato, hops and the like).

Especially preferred are biotrophic pathogens, e.g., Phakopsorapachyrhizi and/or those pathogens which have essentially a similarinfection mechanism as Phakopsora pachyrhizi, as described herein.Particularly preferred are pathogens from the subclass Pucciniomycetes,preferably from the order Pucciniales (rust), previously also known asUredinales, among which in particular the Melompsoraceae. Preferred arePhakopsoraceae, more preferably Phakopsora. Especially preferred arePhakopsora pachyrhizi and/or Phakopsora meibomiae.

Also preferred rust fungi are selected from the group of Puccinia,Gymnosporangium, Juniperus, Cronartium, Hemileia, and Uromyces,preferably Puccinia sorghi, Gymnosporangium juniperi-virginianae,Juniperus virginiana, Cronartium ribicola, Hemlleia vastatrix, Pucciniagraminis, Puccinia coronata, Uromyces phaseoli, Puccinia hemerocallldis,Puccinia persistens subsp. Triticina, Puccinia striiformis, Pucciniagraminis causes, and/or Uromyces appendeculatus.

CASAR Expression Constructs and Vector Constructs

A recombinant nucleic acid, expression cassette or vector constructpreferably comprises a natural gene and a natural promoter, a naturalgene and a non-natural promoter, a non-natural gene and a naturalpromoter, or a non-natural gene and a non-natural promoter.

If protein expression is desired, it is generally desirable to include atranscription termination sequence, e.g., a polyadenylation region atthe 3′-end of a polynucleotide coding region. The polyadenylation regioncan be derived from the natural gene, from a variety of other plantgenes, or from T-DNA. The 3′ end sequence to be added may be derivedfrom, for example, the nopaline synthase or octopine synthase genes, oralternatively from another plant gene, or less preferably from any othereukaryotic gene.

A recombinant vector construct comprising:

-   (a) (i) a nucleic acid having at least 60% identity, preferably at    least 70% sequence identity, at least 80%, at least 90%, at least    95%, at least 98%, at least 99% sequence identity, or even 100%    sequence identity with SEQ ID NO: 2, 1, 5-12, 13, 15, 17, 19, 21,    23, 25, or 27, or a functional fragment thereof, or an orthologue or    a paralogue thereof, or a splice variant thereof;    -   (ii) a nucleic acid coding for a protein having at least 60%        identity, preferably at least 70% sequence identity, at least        80%, at least 90%, at least 95%, at least 98%, at least 99%        sequence identity, or even 100% sequence identity with SEQ ID        NO: 3, 14, 16, 18, 20, 22, 24, 26, or 28, a functional fragment        thereof, an orthologue or a paralogue thereof; preferably the        encoded protein confers enhanced fungal resistance relative to        control plants;    -   (iii) a nucleic acid capable of hybridizing under stringent        conditions with a complementary sequence of any of the nucleic        acids according to (i) or (ii); preferably encoding a CASAR        protein; preferably wherein the nucleic acid molecule codes for        a polypeptide which has essentially identical properties to the        polypeptide described in SEQ ID NO: 3; preferably the encoded        protein confers enhanced fungal resistance relative to control        plants; and/or    -   (iv) a nucleic acid encoding the same CASAR protein as any of        the nucleic acids of (i) to (iii) above, but differing from the        nucleic acids of (i) to (iii) above due to the degeneracy of the        genetic code, operably linked with-   (b) a promoter and-   (c) a transcription termination sequence is a further embodiment of    the invention.

Furthermore, a recombinant vector construct is provided comprising:

-   (a) (i) a nucleic acid having at least 80%, at least 90%, at least    95%, at least 98%, at least 99% sequence identity, or even 100%    sequence identity with SEQ ID NO: 2, 1, 5-12, 13, 15, 17, 19, 21,    23, 25, or 27;    -   (ii) a nucleic acid coding for a protein having at least 80%, at        least 90%, at least 95%, at least 98%, at least 99% sequence        identity, or even 100% sequence identity with SEQ ID NO: 3, 14,        16, 18, 20, 22, 24, 26, or 28; preferably the encoded protein        confers enhanced fungal resistance relative to control plants;    -   (iii) a nucleic acid capable of hybridizing under stringent        conditions with a complementary sequence of any of the nucleic        acids according to (i) or (ii); preferably encoding a CASAR        protein; preferably wherein the nucleic acid molecule codes for        a polypeptide which has essentially identical properties to the        polypeptide described in SEQ ID NO: 3; preferably the encoded        protein confers enhanced fungal resistance relative to control        plants; and/or    -   (iv) a nucleic acid encoding the same CASAR protein as any of        the nucleic acids of (i) to (iii) above, but differing from the        nucleic acids of (i) to (iii) above due to the degeneracy of the        genetic code, operably linked with-   (b) a promoter and-   (c) a transcription termination sequence is a further embodiment of    the invention.

Furthermore, a recombinant vector construct is provided comprising:

-   (a) (i) a nucleic acid having at least 80%, at least 90%, at least    95%, at least 98%, at least 99% sequence identity, or even 100%    sequence identity with SEQ ID NO: 2;    -   (ii) a nucleic acid coding for a protein having at least 80%, at        least 90%, at least 95%, at least 98%, at least 99% sequence        identity, or even 100% sequence identity with SEQ ID NO: 3;        preferably the encoded protein confers enhanced fungal        resistance relative to control plants;    -   (iii) a nucleic acid capable of hybridizing under stringent        conditions with a complementary sequence of any of the nucleic        acids according to (i) or (ii); preferably encoding a CASAR        protein; preferably wherein the nucleic acid molecule codes for        a polypeptide which has essentially identical properties to the        polypeptide described in SEQ ID NO: 3; preferably the encoded        protein confers enhanced fungal resistance relative to control        plants; and/or    -   (iv) a nucleic acid encoding the same CASAR protein as any of        the nucleic acids of (i) to (iii) above, but differing from the        nucleic acids of (i) to (iii) above due to the degeneracy of the        genetic code, operably linked with-   (b) a promoter and-   (c) a transcription termination sequence is a further embodiment of    the invention.

In the case of a genomic library, the natural genetic environment of thenucleic acid sequence is preferably retained, at least in part. Theenvironment preferably flanks the nucleic acid sequence at least on oneside and has a sequence length of at least 50 bp, preferably at least500 bp, especially preferably at least 1000 bp, most preferably at least5000 bp.

Promoters according to the present invention may be constitutive,inducible, in particular pathogen-inducible, developmentalstage-preferred, cell type-preferred, tissue-preferred ororgan-preferred. Preferably, the promoter is a non-natural promoter.Constitutive promoters are active under most conditions. Non-limitingexamples of constitutive promoters include the CaMV 19S and 35Spromoters (Odell et al., 1985, Nature 313:810-812), the sX CaMV 35Spromoter (Kay et al., 1987, Science 236:1299-1302), the Sep1 promoter,the rice actin promoter (McElroy et al., 1990, Plant Cell 2:163-171),the Arabidopsis actin promoter, the ubiquitin promoter (Christensen etal., 1989, Plant Molec. Biol. 18:675-689); pEmu (Last et al., 1991,Theor. Appl. Genet. 81:581-588), the figwort mosaic virus 35S promoter,the Smas promoter (Velten et al., 1984, EMBO J. 3:2723-2730), the GRP1-8promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No.5,683,439), promoters from the T-DNA of Agrobacterium, such as mannopinesynthase, nopaline synthase, and octopine synthase, the small subunit ofribulose biphosphate carboxylase (ssuRUBISCO) promoter, and/or the like.

Preferably, the expression vector of the invention comprises aconstitutive promoter, mesophyll-specific promoter, epidermis-specificpromoter, root-specific promoter, a pathogen inducible promoter, or afungal-inducible promoter.

A promoter is inducible, if its activity, measured on the amount of RNAproduced under control of the promoter, is at least 30%, at least 40%,at least 50% preferably at least 60%, at least 70%, at least 80%, atleast 90% more preferred at least 100%, at least 200%, at least 300%higher in its induced state, than in its un-induced state. A promoter iscell-, tissue- or organ-specific, if its activity, measured on theamount of RNA produced under control of the promoter, is at least 30%,at least 40%, at least 50% preferably at least 60%, at least 70%, atleast 80%, at least 90% more preferred at least 100%, at least 200%, atleast 300% higher in a particular cell-type, tissue or organ, then inother cell-types or tissues of the same plant, preferably the othercell-types or tissues are cell types or tissues of the same plant organ,e.g. a root. In the case of organ specific promoters, the promoteractivity has to be compared to the promoter activity in other plantorgans, e.g. leaves, stems, flowers or seeds. Preferably, the promoteris a constitutive promoter, mesophyll-specific promoter, orepidermis-specific promoter.

In preferred embodiments, the increase in the protein amount and/oractivity of the CASAR protein takes place in a constitutive ortissue-specific manner. In especially preferred embodiments, anessentially pathogen-induced increase in the protein amount and/orprotein activity takes place, for example by recombinant expression ofthe CASAR nucleic acid under the control of a fungal-inducible promoter.In particular, the expression of the CASAR nucleic acid takes place onfungal infected sites, where, however, preferably the expression of theCASAR nucleic acid remains essentially unchanged in tissues not infectedby fungus.

Developmental stage-preferred promoters are preferentially expressed atcertain stages of development. Tissue and organ preferred promotersinclude those that are preferentially expressed in certain tissues ororgans, such as leaves, roots, seeds, or xylem. Examples of tissuepreferred and organ preferred promoters include, but are not limited tofruit-preferred, ovule-preferred, male tissue-preferred, seed-preferred,integument-preferred, tuber-preferred, stalk-preferred,pericarp-preferred, leaf-preferred, stigma-preferred, pollen-preferred,anther-preferred, a petal-preferred, sepal-preferred, pedicel-preferred,silique-preferred, stem-preferred, root-preferred promoters and/or thelike. Seed preferred promoters are preferentially expressed during seeddevelopment and/or germination. For example, seed preferred promoterscan be embryo-preferred, endosperm preferred and seed coat-preferred.See Thompson et al., 1989, BioEssays 10:108. Examples of seed preferredpromoters include, but are not limited to cellulose synthase (celA),Cim1, gamma-zein, globulin-1, maize 19 kD zein (cZ19B1) and/or the like.

Other suitable tissue-preferred or organ-preferred promoters include,but are not limited to, the napin-gene promoter from rapeseed (U.S. Pat.No. 5,608,152), the USP-promoter from Vicia faba (Baeumlein et al.,1991, Mol Gen Genet. 225(3):459-67), the oleosin-promoter fromArabidopsis (PCT Application No. WO 98/45461), the phaseolin-promoterfrom Phaseolus vulgaris (U.S. Pat. No. 5,504,200), the Bce4-promoterfrom Brassica (PCT Application No. WO 91/13980), or the legumin B4promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2(2):233-9), aswell as promoters conferring seed specific expression in monocot plantslike maize, barley, wheat, rye, rice, etc. Suitable promoters to noteare the Ipt2 or Ipt1-gene promoter from barley (PCT Application No. WO95/15389 and PCT Application No. WO 95/23230) or those described in PCTApplication No. WO 99/16890 (promoters from the barley hordein-gene,rice glutelin gene, rice oryzin gene, rice prolamin gene, wheat gliadingene, wheat glutelin gene, oat glutelin gene, Sorghum kasirin-gene,and/or rye secalin gene).

Promoters useful according to the invention include, but are not limitedto, are the major chlorophyll a/b binding protein promoter, histonepromoters, the Ap3 promoter, the β-conglycin promoter, the napinpromoter, the soybean lectin promoter, the maize 15 kD zein promoter,the 22 kD zein promoter, the 27 kD zein promoter, the g-zein promoter,the waxy, shrunken 1, shrunken 2, bronze promoters, the Zm13 promoter(U.S. Pat. No. 5,086,169), the maize polygalacturonase promoters (PG)(U.S. Pat. Nos. 5,412,085 and 5,545,546), the SGB6 promoter (U.S. Pat.No. 5,470,359), as well as synthetic or other natural promoters.

Epidermis-specific promoters may be selected from the group consistingof:

WIR5 (=GstA1); acc. X56012; Dudler & Schweizer,GLP4, acc. AJ310534; Wei Y., Zhang Z., Andersen C. H., Schmelzer E.,Gregersen P. L., Collinge D. B., Smedegaard-Petersen V. andThordal-Christensen H., Plant Molecular Biology 36, 101 (1998),GLP2a, acc. AJ237942, Schweizer P., Christoffel A. and Dudler R., PlantJ. 20, 541 (1999); Prx7, acc. AJ003141, Kristensen B. K., Ammitzböll H.,Rasmussen S. K. and Nielsen K. A., Molecular Plant Pathology, 2(6), 311(2001);GerA, acc. AF250933; Wu S., Druka A., Horvath H., Kleinhofs A.,Kannangara G. and von Wettstein D., Plant Phys Biochem 38, 685 (2000);OsROC1, acc. AP004656RTBV, acc. AAV62708, AAV62707; Klöti A., Henrich C., Bieri S., He X.,Chen G., Burkhardt P. K., Wünn J., Lucca P., Hohn T., Potrykus I. andFütterer J., PMB 40, 249 (1999);Chitinase ChtC2-Promoter from potato (Ancillo et al., Planta. 217(4),566, (2003));

AtProT3 Promoter (Grallath et al., Plant Physiology. 137(1), 117(2005));

SHN-Promoters from Arabidopsis (AP2/EREBP transcription factors involvedin cutin and wax production) (Aarón et al., Plant Cell. 16(9), 2463(2004)); and/orGSTA1 from wheat (Dudler et al., WP2005306368 and Altpeter et al., PlantMolecular Biology. 57(2), 271 (2005)).

Mesophyll-specific promoters may be selected from the group consistingof:

PPCZm1 (=PEPC); Kausch A. P., Owen T. P., Zachwieja S. J., Flynn A. R.and Sheen J., Plant Mol. Biol. 45, 1 (2001);OsrbcS, Kyozuka et al., PlaNT Phys 102, 991 (1993); Kyozuka J., McElroyD., Hayakawa T., Xie Y., Wu R. and Shimamoto K., Plant Phys. 102, 991(1993); OsPPDK, acc. AC099041;TaGF-2.8, acc. M63223; Schweizer P., Christoffel A. and Dudler R., PlantJ. 20, 541 (1999);TaFBPase, acc. X53957;TaWIS1, acc. AF467542; US 200220115849;HvBIS1, acc. AF467539; US 200220115849;ZmMIS1, acc. AF467514; US 200220115849;HvPR1a, acc. X74939; Bryngelsson et al., Mol. Plant Microbe Interacti. 7(2), 267 (1994);HvPR1 b, acc. X74940; Bryngelsson et al., Mol. Plant Microbe Interact.7(2), 267 (1994);HvB1,3gluc; acc. AF479647;HvPrx8, acc. AJ276227; Kristensen et al., Molecular Plant Pathology,2(6), 311 (2001); and/orHvPAL, acc. X97313; Wei Y., Zhang Z., Andersen C. H., Schmelzer E.,Gregersen P. L., Collinge D. B., Smedegaard-Petersen V. andThordal-Christensen H. Plant Molecular Biology 36, 101 (1998)Rust induced mesophyll specific promoter 820, as shown in SEQ ID NO: 4.

Constitutive promoters may be selected from the group consisting of:

-   PcUbi promoter from parsley (WO 03/102198)-   CaMV 35S promoter: Cauliflower Mosaic Virus 35S promoter (Benfey et    al. 1989 EMBO J. 8(8): 2195-2202),-   STPT promoter: Arabidopsis thaliana Short Triose phosphate    translocator promoter (Accession NM_123979)-   Act1 promoter: Oryza sativa actin 1 gene promoter (McElroy et al.    1990 PLANT CELL 2(2) 163-171 a) and/or-   EF1A2 promoter: Glycine max translation elongation factor EF1 alpha    (US 20090133159).

In preferred embodiments, the increase in the protein quantity orfunction of the CASAR protein takes place in a constitutive ortissue-specific manner. In especially preferred embodiments, anessentially pathogen-induced increase in the protein quantity or proteinfunction takes place, for example by exogenous expression of the CASARnucleic acid under the control of a fungal-inducible promoter,preferably a rust-inducible promoter. In particular, the expression ofthe CASAR nucleic acid takes place on fungal infected sites, where,however, preferably the expression of the CASAR nucleic acid sequenceremains essentially unchanged in tissues not infected by fungus.

Preferably, the CASAR nucleic acid is under the control of a rustinduced mesophyll specific promoter. More preferably, the promoter isthe rust induced mesophyll specific promoter 820, preferably, as shownin SEQ ID NO: 4. Preferably, the rust induced mesophyll specificpromoter comprises a nucleic acid sequence which is at least 70%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, or atleast 99% identical with the nucleic acid sequence shown in SEQ ID NO:4.

A preferred terminator is the terminator of the cathepsin D inhibitorgene from Solanum tuberosum.

Preferred promoter-terminator combinations with the gene of interest inbetween are a promoter from parsley, preferably, the parsley ubiquitinepromoter, in combination with the terminator of the cathepsin Dinhibitor gene from Solanum tuberosum. Another preferredpromoter-terminator combination is the rust induced mesophyll specificpromoter 820 in combination with the terminator of the cathepsin Dinhibitor gene from Solanum tuberosum.

An intron sequence may also be added to the 5′ untranslated region (UTR)and/or the coding sequence of the partial coding sequence to increasethe amount of the mature message that accumulates in the cytosol.Inclusion of a spliceable intron in the transcription unit in both plantand animal expression constructs has been shown to increase geneexpression at both the mRNA and protein levels up to 1000-fold (Buchmanand Berg (1988) Mol. Cell biol. 8: 4395-4405; Callis et al. (1987) GenesDev 1:1183-1200). Such intron enhancement of gene expression istypically greatest when placed near the 5′ end of the transcriptionunit. Use of the maize introns Adh1-S intron 1, 2, and 6, the Bronze-1intron are known in the art. For general information see: The MaizeHandbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).

One type of vector construct is a “plasmid,” which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectorconstructs are capable of autonomous replication in a host plant cellinto which they are introduced. Other vector constructs are integratedinto the genome of a host plant cell upon introduction into the hostcell, and thereby are replicated along with the host genome. Inparticular the vector construct is capable of directing the expressionof gene to which the vectors is operatively linked. However, theinvention is intended to include such other forms of expression vectorconstructs, such as viral vectors (e.g., potato virus X, tobacco rattlevirus, and/or Gemini virus), which serve equivalent functions.

Transgenic Organisms; Transgenic Plants, Plant Parts, and Plant Cells

A preferred embodiment is a transgenic plant, transgenic plant part, ortransgenic plant cell overexpressing an exogenous CASAR protein.Preferably, the CASAR protein overexpressed in the plant, plant part orplant cell is encoded by

-   (i) an exogenous nucleic acid having at least 60% identity with SEQ    ID NO: 2, 1, 5-12, 13, 15, 17, 19, 21, 23, 25, or 27, or a    functional fragment thereof, an orthologue or a paralogue thereof,    or a splice variant thereof; or by-   (ii) an exogenous nucleic acid encoding a protein having at least    60% identity with SEQ ID NO: 3, 14, 16, 18, 20, 22, 24, 26, or 28, a    functional fragment thereof, an orthologue or a paralogue thereof;    preferably the encoded protein confers enhanced fungal resistance    relative to control plants;-   (iii) an exogenous nucleic acid capable of hybridizing under    stringent conditions with a complementary sequence of any of the    nucleic acids according to (i) or (ii); preferably encoding a CASAR    protein; preferably wherein the nucleic acid molecule codes for a    polypeptide which has essentially identical properties to the    polypeptide described in SEQ ID NO: 3; preferably the encoded    protein confers enhanced fungal resistance relative to control    plants; and/or by-   (iv) an exogenous nucleic acid encoding the same CASAR protein as    any of the nucleic acids of (i) to (iii) above, but differing from    the nucleic acids of (i) to (iii) above due to the degeneracy of the    genetic code.

Most preferably, the exogenous nucleic acid has at least 80%, at least90%, at least 95%, at least 98%, at least 99% sequence identity, or even100% sequence identity with SEQ ID NO: 2; or comprises an exogenousnucleic acid encoding a protein having at least 80%, at least 90%, atleast 95%, at least 98%, at least 99% sequence identity, or even 100%sequence identity with SEQ ID NO: 3.

A preferred embodiment is a transgenic plant, transgenic plant part, ortransgenic plant cell overexpressing an exogenous CASAR protein.Preferably, the CASAR protein overexpressed in the plant, plant part orplant cell is encoded by

-   (i) an exogenous nucleic acid having at least 60% identity with SEQ    ID NO: 2 or a functional fragment thereof, an orthologue or a    paralogue thereof, or a splice variant thereof; or by-   (ii) an exogenous nucleic acid encoding a protein having at least    60% identity with SEQ ID NO: 3, a functional fragment thereof, an    orthologue or a paralogue thereof; preferably the encoded protein    confers enhanced fungal resistance relative to control plants;-   (iii) an exogenous nucleic acid capable of hybridizing under    stringent conditions with a complementary sequence of any of the    nucleic acids according to (i) or (ii); preferably encoding a CASAR    protein; preferably wherein the nucleic acid molecule codes for a    polypeptide which has essentially identical properties to the    polypeptide described in SEQ ID NO: 3; preferably the encoded    protein confers enhanced fungal resistance relative to control    plants; and/or by-   (iv) an exogenous nucleic acid encoding the same CASAR protein as    any of the nucleic acids of (i) to (iii) above, but differing from    the nucleic acids of (i) to (iii) above due to the degeneracy of the    genetic code.

Most preferably, the exogenous nucleic acid has at least 80%, at least90%, at least 95%, at least 98%, at least 99% sequence identity, or even100% sequence identity with SEQ ID NO: 2; or comprises an exogenousnucleic acid encoding a protein having at least 95%, at least 98%, atleast 99% sequence identity, or even 100% sequence identity with SEQ IDNO: 3.

In preferred embodiments, the protein amount of a CASAR protein in thetransgenic plant is increased by at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, or at least 95% or more in comparison to a wild typeplant that is not transformed with the CASAR nucleic acid.

More preferably, the transgenic plant, transgenic plant part, ortransgenic plant cell according to the present invention has beenobtained by transformation with a recombinant vector described herein.

Suitable methods for transforming or transfecting host cells includingplant cells are well known in the art of plant biotechnology. Any methodmay be used to transform the recombinant expression vector into plantcells to yield the transgenic plants of the invention. General methodsfor transforming dicotyledonous plants are disclosed, for example, inU.S. Pat. Nos. 4,940,838; 5,464,763, and the like. Methods fortransforming specific dicotyledonous plants, for example, cotton, areset forth in U.S. Pat. Nos. 5,004,863; 5,159,135; and 5,846,797. Soytransformation methods are set forth in U.S. Pat. Nos. 4,992,375;5,416,011; 5,569,834; 5,824,877; 6,384,301 and in EP 0301749B1 may beused. Transformation methods may include direct and indirect methods oftransformation. Suitable direct methods include polyethylene glycolinduced DNA uptake, liposome-mediated transformation (U.S. Pat. No.4,536,475), biolistic methods using the gene gun (Fromm M E et al.,Bio/Technology. 8(9):833-9, 1990; Gordon-Kamm et al. Plant Cell 2:603,1990), electroporation, incubation of dry embryos in DNA-comprisingsolution, and microinjection. In the case of these direct transformationmethods, the plasmids used need not meet any particular requirements.Simple plasmids, such as those of the pUC series, pBR322, M13mp series,pACYC184 and the like can be used. If intact plants are to beregenerated from the transformed cells, an additional selectable markergene is preferably located on the plasmid. The direct transformationtechniques are equally suitable for dicotyledonous and monocotyledonousplants.

Transformation can also be carried out by bacterial infection by meansof Agrobacterium (for example EP 0 116 718), viral infection by means ofviral vectors (EP 0 067 553; U.S. Pat. No. 4,407,956; WO 95/34668; WO93/03161) or by means of pollen (EP 0 270 356; WO 85/01856; U.S. Pat.No. 4,684,611). Agrobacterium based transformation techniques(especially for dicotyledonous plants) are well known in the art. TheAgrobacterium strain (e.g., Agrobacterium tumefaciens or Agrobacteriumrhizogenes) comprises a plasmid (Ti or Ri plasmid) and a T-DNA elementwhich is transferred to the plant following infection withAgrobacterium. The T-DNA (transferred DNA) is integrated into the genomeof the plant cell. The T-DNA may be localized on the Ri- or Ti-plasmidor is separately comprised in a so-called binary vector. Methods for theAgrobacterium-mediated transformation are described, for example, inHorsch R B et al. (1985) Science 225:1229. The Agrobacterium-mediatedtransformation is best suited to dicotyledonous plants but has also beenadapted to monocotyledonous plants. The transformation of plants byAgrobacteria is described in, for example, White F F, Vectors for GeneTransfer in Higher Plants, Transgenic Plants, Vol. 1, Engineering andUtilization, edited by S. D. Kung and R. Wu, Academic Press, 1993, pp.15-38; Jenes B et al. Techniques for Gene Transfer, Transgenic Plants,Vol. 1, Engineering and Utilization, edited by S. D. Kung and R. Wu,Academic Press, 1993, pp. 128-143; Potrykus (1991) Annu Rev PlantPhysiol Plant Molec Biol 42:205-225. Transformation may result intransient or stable transformation and expression. Although a nucleotidesequence of the present invention can be inserted into any plant andplant cell falling within these broad classes, it is particularly usefulin crop plant cells.

The genetically modified plant cells can be regenerated via all methodswith which the skilled worker is familiar. Suitable methods can be foundin the abovementioned publications by S. D. Kung and R. Wu, Potrykus orHöfgen and Willmitzer.

After transformation, plant cells or cell groupings may be selected forthe presence of one or more markers which are encoded byplant-expressible genes co-transferred with the gene of interest,following which the transformed material is regenerated into a wholeplant. To select transformed plants, the plant material obtained in thetransformation is, as a rule, subjected to selective conditions so thattransformed plants can be distinguished from untransformed plants. Forexample, the seeds obtained in the above-described manner can be plantedand, after an initial growing period, subjected to a suitable selectionby spraying. A further possibility consists in growing the seeds, ifappropriate after sterilization, on agar plates using a suitableselection agent so that only the transformed seeds can grow into plants.Alternatively, the transformed plants are screened for the presence of aselectable marker such as the ones described above. The transformedplants may also be directly selected by screening for the presence ofthe CASAR nucleic acid.

Following DNA transfer and regeneration, putatively transformed plantsmay also be evaluated, for instance using Southern analysis, for thepresence of the gene of interest, copy number and/or genomicorganisation. Alternatively or additionally, expression levels of thenewly introduced DNA may be monitored using Northern and/or Westernanalysis, both techniques being well known to persons having ordinaryskill in the art.

The generated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedand homozygous second-generation (or T2) transformants selected, and theT2 plants may then further be propagated through classical breedingtechniques. The generated transformed organisms may take a variety offorms. For example, they may be chimeras of transformed cells andnon-transformed cells; clonal transformants (e.g., all cells transformedto contain the expression cassette); grafts of transformed anduntransformed tissues (e.g., in plants, a transformed rootstock graftedto an untransformed scion).

Preferably, constructs or vectors or expression cassettes are notpresent in the genome of the original plant or are present in the genomeof the transgenic plant not at their natural locus of the genome of theoriginal plant.

Preferably, the transgenic plant of the present invention or the plantobtained by the method of the present invention has increased resistanceagainst fungal pathogens, preferably rust pathogens (i.e., fungalpathogens of the order Pucciniales), preferably against fungal pathogensof the family Phakopsoraceae, more preferably against fungal pathogensof the genus Phakopsora, most preferably against Phakopsora pachyrhiziand Phakopsora meibomiae, also known as soybean rust. Preferably,resistance against Phakopsora pachyrhizi and/or Phakopsora meibomiae isincreased.

Preferably, the plant, plant part, or plant cell is a plant or derivedfrom a plant selected from the group consisting of beans, soya, pea,clover, kudzu, lucerne, lentils, lupins, vetches, groundnut, rice,wheat, barley, Arabidopsis, lentil, banana, canola, cotton, potato,corn, sugar cane, alfalfa, and sugar beet.

In one embodiment of the present invention the plant is selected fromthe group consisting of beans, soya, pea, clover, kudzu, lucerne,lentils, lupins, vetches, and/or groundnut. Preferably, the plant is alegume, comprising plants of the genus Phaseolus (comprising Frenchbean, dwarf bean, climbing bean (Phaseolus vulgaris), Lima bean(Phaseolus lunatus L.), Tepary bean (Phaseolus acutifolius A. Gray),runner bean (Phaseolus coccineus)); the genus Glycine (comprisingGlycine soja, soybeans (Glycine max (L.) Merill)); pea (Pisum)(comprising shelling peas (Pisum sativum L. convar. sativum), alsocalled smooth or round-seeded peas; marrowfat pea (Pisum sativum L.convar. medullare Alef. emend. C.O. Lehm), sugar pea (Pisum sativum L.convar. axiphium Alef emend. C.O. Lehm), also called snow pea,edible-podded pea or mangetout, (Pisum granda sneida L. convar. sneidulop. shneiderium)); peanut (Arachis hypogaea), clover (Trifolium spec.),medick (Medicago), kudzu vine (Pueraria lobata), common lucerne, alfalfa(M. sativa L.), chickpea (Cicer), lentils (Lens) (Lens culinarisMedik.), lupins (Lupinus); vetches (Vicia), field bean, broad bean(Vicia faba), vetchling (Lathyrus) (comprising chickling pea (Lathyrussativus), heath pea (Lathyrus tuberosus)); genus Vigna (comprising mothbean (Vigna aconitifolia (Jacq.) Maréchal), adzuki bean (Vigna angularis(Willd.) Ohwi & H. Ohashi), urd bean (Vigna mungo (L.) Hepper), mungbean (Vigna radiata (L.) R. Wilczek), bambara groundnut (Vignasubterrane (L.) Verdc.), rice bean (Vigna umbellata (Thunb.) Ohwi & H.Ohashi), Vigna vexillata (L.) A. Rich., Vigna unguiculata (L.) Walp., inthe three subspecies asparagus bean, cowpea, catjang bean)); pigeonpea(Cajanus cajan (L.) Millsp.), the genus Macrotyloma (comprising geocarpagroundnut (Macrotyloma geocarpum (Harms) Maréchal & Baudet), horse bean(Macrotyloma uniflorum (Lam.) Verdc.); goa bean (Psophocarpustetragonolobus (L.) DC.), African yam bean (Sphenostylis stenocarpa(Hochst. ex A. Rich.) Harms), Egyptian black bean, dolichos bean, lablabbean (Lablab purpureus (L.) Sweet), yam bean (Pachyrhizus), guar bean(Cyamopsis tetragonolobus (L.) Taub.); and/or the genus Canavalia(comprising jack bean (Canavalia ensiformis (L.) DC.), sword bean(Canavalia gladiata (Jacq.) DC.).

Further preferred is a plant selected from the group consisting ofbeans, soya, pea, clover, kudzu, lucerne, lentils, lupins, vetches, andgroundnut. Most preferably, the plant, plant part, or plant cell is oris derived from soy.

Preferably, the transgenic plant of the present invention or the plantobtained by the method of the present invention is a soybean plant andhas increased resistance against fungal pathogens of the orderPucciniales (rust), preferably, of the family Phakopsoraceae, morepreferably against fungal pathogens of the genus Phakopsora, mostpreferably against Phakopsora pachyrhizi and Phakopsora meibomiae, alsoknown as soybean rust. Preferably, resistance against Phakopsorapachyrhizi and/or Phakopsora meibomiae is increased.

Methods for the Production of Transgenic Plants

One embodiment according to the present invention provides a method forproducing a transgenic plant, a transgenic plant part, or a transgenicplant cell resistant to a fungal pathogen, preferably of the familyPhakopsoraceae, for example soybean rust, wherein the recombinantnucleic acid used to generate a transgenic plant comprises a promoterthat is functional in the plant cell, operably linked to a CASAR nucleicacid, which is preferably SEQ ID NO: 2, 1, 5-12, 13, 15, 17, 19, 21, 23,25, or 27, and

a terminator regulatory sequence.

In one embodiment, the present invention refers to a method for theproduction of a transgenic plant, transgenic plant part, or transgenicplant cell having increased fungal resistance, comprising

-   (a) introducing a recombinant vector construct according to the    present invention into a plant, a plant part or a plant cell and-   (b) generating a transgenic plant from the plant, plant part or    plant cell.

Preferably, the method for the production of the transgenic plant,transgenic plant part, or transgenic plant cell further comprises thestep

-   (c) expressing the CASAR protein, preferably encoded by    -   (i) an exogenous nucleic acid having at least 60% identity with        SEQ ID NO: 2, 1, 5-12, 13, 15, 17, 19, 21, 23, 25, or 27, a        functional fragment thereof, an orthologue or a paralogue        thereof, or a splice variant thereof;    -   (ii) an exogenous nucleic acid encoding a protein having at        least 60% identity with SEQ ID NO: 3, 14, 16, 18, 20, 22, 24,        26, or 28, or a functional fragment thereof, an orthologue or a        paralogue thereof; preferably the encoded protein confers        enhanced fungal resistance relative to control plants;    -   (iii) an exogenous nucleic acid capable of hybridizing under        stringent conditions with a complementary sequence of any of the        nucleic acids according to (i) or (ii); preferably encoding a        CASAR protein; preferably wherein the nucleic acid molecule        codes for a polypeptide which has essentially identical        properties to the polypeptide described in SEQ ID NO: 3;        preferably the encoded protein confers enhanced fungal        resistance relative to control plants; and/or by    -   (iv) an exogenous nucleic acid encoding the same CASAR protein        as any of the nucleic acids of (i) to (iii) above, but differing        from the nucleic acids of (i) to (iii) above due to the        degeneracy of the genetic code.

Preferably, said introducing and expressing does not comprise anessentially biological process.

More preferably, the method for the production of the transgenic plant,transgenic plant part, or transgenic plant cell further comprises thestep

-   (c) expressing the CASAR protein, preferably encoded by    -   (i) an exogenous nucleic acid having at least 60% identity with        SEQ ID NO: 2, a functional fragment thereof, an orthologue or a        paralogue thereof, or a splice variant thereof;    -   (ii) an exogenous nucleic acid encoding a protein having at        least 60% identity with SEQ ID NO: 3, or a functional fragment        thereof, an orthologue or a paralogue thereof; preferably the        encoded protein confers enhanced fungal resistance relative to        control plants;    -   (iii) an exogenous nucleic acid capable of hybridizing under        stringent conditions with a complementary sequence of any of the        nucleic acids according to (i) or (ii); preferably encoding a        CASAR protein; preferably wherein the nucleic acid molecule        codes for a polypeptide which has essentially identical        properties to the polypeptide described in SEQ ID NO: 3;        preferably the encoded protein confers enhanced fungal        resistance relative to control plants; and/or by    -   (iv) an exogenous nucleic acid encoding the same CASAR protein        as any of the nucleic acids of (i) to (iii) above, but differing        from the nucleic acids of (i) to (iii) above due to the        degeneracy of the genetic code.

Preferably, the method for the production of the transgenic plant,transgenic plant part, or transgenic plant cell further comprises thestep of selecting a transgenic plant expressing

-   (i) an exogenous nucleic acid having at least 60% identity,    preferably at least 70% sequence identity, at least 80%, at least    90%, at least 95%, at least 98%, at least 99% sequence identity, or    even 100% sequence identity with SEQ ID NO: 2, 1, 5-12, 13, 15, 17,    19, 21, 23, 25, or 27, or a functional fragment thereof, or an    orthologue or a paralogue thereof, or a splice variant thereof;-   (ii) an exogenous nucleic acid coding for a protein having at least    60% identity, preferably at least 70% sequence identity, at least    80%, at least 90%, at least 95%, at least 98%, at least 99% sequence    identity, or even 100% sequence identity with SEQ ID NO: 3, 14, 16,    18, 20, 22, 24, 26, or 28, a functional fragment thereof, an    orthologue or a paralogue thereof; preferably the encoded protein    confers enhanced fungal resistance relative to control plants;-   (iii) an exogenous nucleic acid capable of hybridizing under    stringent conditions with a complementary sequence of any of the    nucleic acids according to (i) or (ii); preferably encoding a CASAR    protein; preferably wherein the nucleic acid molecule codes for a    polypeptide which has essentially identical properties to the    polypeptide described in SEQ ID NO: 3; preferably the encoded    protein confers enhanced fungal resistance relative to control    plants; and/or-   (iv) an exogenous nucleic acid encoding the same CASAR polypeptide    as any of the nucleic acids of (i) to (iii) above, but differing    from the nucleic acids of (i) to (iii) above due to the degeneracy    of the genetic code.

Preferably, the method for the production of the transgenic plant,transgenic plant part, or transgenic plant cell additionally comprisesthe step of harvesting the seeds of the transgenic plant and plantingthe seeds and growing the seeds to plants, wherein the grown plant(s)comprises

-   (i) the exogenous nucleic acid having at least 60% identity with SEQ    ID NO: 2, 1, 5-12, 13, 15, 17, 19, 21, 23, 25, or 27, a functional    fragment thereof, an orthologue or a paralogue thereof, or a splice    variant thereof;-   (ii) the exogenous nucleic acid encoding a protein having at least    60% identity with SEQ ID NO: 3, 14, 16, 18, 20, 22, 24, 26, or 28,    or a functional fragment thereof, an orthologue or a paralogue    thereof; preferably the encoded protein confers enhanced fungal    resistance relative to control plants;-   (iii) the exogenous nucleic acid capable of hybridizing under    stringent conditions with a complementary sequence of any of the    nucleic acids according to (i) or (ii); preferably encoding a CASAR    protein; preferably wherein the nucleic acid molecule codes for a    polypeptide which has essentially identical properties to the    polypeptide described in SEQ ID NO: 3; preferably the encoded    protein confers enhanced fungal resistance relative to control    plants; and/or-   (iv) the exogenous nucleic acid encoding the same CASAR protein as    any of the nucleic acids of (i) to (iii) above, but differing from    the nucleic acids of (i) to (iii) above due to the degeneracy of the    genetic code;    preferably, the step of harvesting the seeds of the transgenic plant    and planting the seeds and growing the seeds to plants, wherein the    grown plant(s) comprises-   (i) the exogenous nucleic acid having at least 60% identity with SEQ    ID NO: 2, 1, 5-12, 13, 15, 17, 19, 21, 23, 25, or 27, a functional    fragment thereof, an orthologue or a paralogue thereof, or a splice    variant thereof;-   (ii) the exogenous nucleic acid encoding a protein having at least    60% identity with SEQ ID NO: 3, 14, 16, 18, 20, 22, 24, 26, or 28,    or a functional fragment thereof, an orthologue or a paralogue    thereof; preferably the encoded protein confers enhanced fungal    resistance relative to control plants;-   (iii) the exogenous nucleic acid capable of hybridizing under    stringent conditions with a complementary sequence of any of the    nucleic acids according to (i) or (ii); preferably encoding a CASAR    protein; preferably wherein the nucleic acid molecule codes for a    polypeptide which has essentially identical properties to the    polypeptide described in SEQ ID NO: 3; preferably the encoded    protein confers enhanced fungal resistance relative to control    plants; and/or-   (iv) the exogenous nucleic acid encoding the same CASAR protein as    any of the nucleic acids of (i) to (iii) above, but differing from    the nucleic acids of (i) to (iii) above due to the degeneracy of the    genetic code;    is repeated more than one time, preferably, 1, 2, 3, 4, 5, 6, 7, 8,    9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times.

The transgenic plants may be selected by known methods as describedabove (e.g., by screening for the presence of one or more markers whichare encoded by plant-expressible genes co-transferred with the CASARgene or by directly screening for the CASAR nucleic acid).

Furthermore, the use of the exogenous CASAR nucleic acid or therecombinant vector construct comprising the CASAR nucleic acid for thetransformation of a plant, plant part, or plant cell to provide a fungalresistant plant, plant part, or plant cell is provided.

Harvestable Parts and Products

Harvestable parts of the transgenic plant according to the presentinvention are part of the invention. Preferably, the harvestable partscomprise the CASAR nucleic acid or CASAR protein. The harvestable partsmay be seeds, roots, leaves and/or flowers comprising the CASAR nucleicacid or CASAR protein or parts thereof. Preferred parts of soy plantsare soy beans comprising the CASAR nucleic acid or CASAR protein.

Products derived from a transgenic plant according to the presentinvention, parts thereof or harvestable parts thereof are part of theinvention. A preferred product is meal or oil, preferably, soybean mealor soybean oil. Preferably, the soybean meal and/or oil comprises theCASAR nucleic acid or CASAR protein.

Preferably the harvestable parts of the transgenic plant according tothe present invention or the products derived from a transgenic plantcomprise an exogenous nucleic acid molecule consisting of or comprisinga nucleic acid selected from the group consisting of:

-   (i) an exogenous nucleic acid having in increasing order of    preference at least at least 70%, at least 71%, at least 72%, at    least 73%, at least 74%, at least 75%, at least 76%, at least 77%,    at least 78%, at least 79%, at least 80%, at least 81%, at least    82%, at least 83%, at least 84%, at least 85%, at least 86%, at    least 87%, at least 88%, at least 89%, at least 90%, at least 91%,    at least 92%, at least 93%, at least 94%, at least 95%, at least    96%, at least 97%, at least 98%, at least 99% or 100% sequence    identity to the nucleic acid sequence represented by SEQ ID NO: 2,    1, 5-12, 13, 15, 17, 19, 21, 23, 25, or 27, or a functional    fragment, derivative, orthologue, or paralogue thereof, or a splice    variant thereof;-   (ii) an exogenous nucleic acid encoding a CASAR protein comprising    an amino acid sequence having in increasing order of preference at    least 70%, at least 71%, at least 72%, at least 73%, at least 74%,    at least 75%, at least 76%, at least 77%, at least 78%, at least    79%, at least 80%, at least 81%, at least 82%, at least 83%, at    least 84%, at least 85%, at least 86%, at least 87%, at least 88%,    at least 89%, at least 90%, at least 91%, at least 92%, at least    93%, at least 94%, at least 95%, at least 96%, at least 97%, at    least 98%, at least 99% or 100% sequence identity to the amino acid    sequence represented by SEQ ID NO: 3, 14, 16, 18, 20, 22, 24, 26, or    28, or a functional fragment, derivative, orthologue, or paralogue    thereof; preferably the CASAR protein has essentially the same    biological activity as a CASAR protein encoded by SEQ ID NO: 2 or 1;    preferably the CASAR protein confers enhanced fungal, preferably    rust, resistance relative to control plants;-   (iii) an exogenous nucleic acid molecule which hybridizes with a    complementary sequence of any of the nucleic acid molecules of (i)    or (ii) under high stringency hybridization conditions; preferably    encoding a CASAR protein; preferably wherein the nucleic acid    molecule codes for a polypeptide which has essentially identical    properties to the polypeptide described in SEQ ID NO: 3; preferably    the encoded protein confers enhanced fungal, preferably rust,    resistance relative to control plants; and-   (iv) an exogenous nucleic acid encoding the same CASAR protein as    the CASAR nucleic acids of (i) to (iii) above, but differing from    the CASAR nucleic acids of (i) to (iii) above due to the degeneracy    of the genetic code,    or wherein the harvestable part of the transgenic plant or the    product derived from the transgenic plant comprises a CASAR protein    encoded by any one of the CASAR nucleic acids of (i) to (iv).

Methods for Manufacturing a Product

The plants, plant parts or plant cells of the invention or obtainable bythe methods of invention can be used for the manufacturing of aharvestable part or product.

In one embodiment the method for the production of a product comprises

-   a) growing the plants of the invention or obtainable by the methods    of invention and-   b) producing said product from or by the plants of the invention    and/or parts, e.g. seeds, of these plants.

In a further embodiment the method comprises the steps

a) growing the plants of the invention,b) removing the harvestable parts as defined above from the plants andc) producing said product from or by the harvestable parts of theinvention.

Preferably the products obtained by said method comprises an exogenousnucleic acid molecule consisting of or comprising a nucleic acidselected from the group consisting of:

-   (i) an exogenous nucleic acid having in increasing order of    preference at least at least 70%, at least 71%, at least 72%, at    least 73%, at least 74%, at least 75%, at least 76%, at least 77%,    at least 78%, at least 79%, at least 80%, at least 81%, at least    82%, at least 83%, at least 84%, at least 85%, at least 86%, at    least 87%, at least 88%, at least 89%, at least 90%, at least 91%,    at least 92%, at least 93%, at least 94%, at least 95%, at least    96%, at least 97%, at least 98%, at least 99% or 100% sequence    identity to the nucleic acid sequence represented by SEQ ID NO: 2,    1, 5-12, 13, 15, 17, 19, 21, 23, 25, or 27, or a functional    fragment, derivative, orthologue, or paralogue thereof, or a splice    variant thereof;-   (ii) an exogenous nucleic acid encoding a CASAR protein comprising    an amino acid sequence having in increasing order of preference at    least 70%, at least 71%, at least 72%, at least 73%, at least 74%,    at least 75%, at least 76%, at least 77%, at least 78%, at least    79%, at least 80%, at least 81%, at least 82%, at least 83%, at    least 84%, at least 85%, at least 86%, at least 87%, at least 88%,    at least 89%, at least 90%, at least 91%, at least 92%, at least    93%, at least 94%, at least 95%, at least 96%, at least 97%, at    least 98%, at least 99% or 100% sequence identity to the amino acid    sequence represented by SEQ ID NO: 3, 14, 16, 18, 20, 22, 24, 26, or    28, or a functional fragment, derivative, orthologue, or paralogue    thereof; preferably the CASAR protein has essentially the same    biological activity as a CASAR protein encoded by SEQ ID NO: 2 or 1;    preferably the CASAR protein confers enhanced fungal, preferably    rust, resistance relative to control plants;-   (iii) an exogenous nucleic acid molecule which hybridizes with a    complementary sequence of any of the nucleic acid molecules of (i)    or (ii) under high stringency hybridization conditions; preferably    encoding a CASAR protein; preferably wherein the nucleic acid    molecule codes for a polypeptide which has essentially identical    properties to the polypeptide described in SEQ ID NO: 3; preferably    the encoded protein confers enhanced fungal, preferably rust,    resistance relative to control plants; and-   (iv) an exogenous nucleic acid encoding the same CASAR protein as    the CASAR nucleic acids of (i) to (iii) above, but differing from    the CASAR nucleic acids of (i) to (iii) above due to the degeneracy    of the genetic code,    or wherein the product obtained by said method comprises a CASAR    protein encoded by any one of the CASAR nucleic acids of (i) to    (iv).

The product may be produced at the site where the plant has been grown,the plants and/or parts thereof may be removed from the site where theplants have been grown to produce the product. Typically, the plant isgrown, the desired harvestable parts are removed from the plant, iffeasible in repeated cycles, and the product made from the harvestableparts of the plant. The step of growing the plant may be performed onlyonce each time the methods of the invention is performed, while allowingrepeated times the steps of product production e.g. by repeated removalof harvestable parts of the plants of the invention and if necessaryfurther processing of these parts to arrive at the product. It is alsopossible that the step of growing the plants of the invention isrepeated and plants or harvestable parts are stored until the productionof the product is then performed once for the accumulated plants orplant parts. Also, the steps of growing the plants and producing theproduct may be performed with an overlap in time, even simultaneously toa large extend or sequentially. Generally the plants are grown for sometime before the product is produced.

In one embodiment the products produced by said methods of the inventionare plant products such as, but not limited to, a foodstuff, feedstuff,a food supplement, feed supplement, fiber, cosmetic and/orpharmaceutical. Foodstuffs are regarded as compositions used fornutrition and/or for supplementing nutrition. Animal feedstuffs andanimal feed supplements, in particular, are regarded as foodstuffs.

In another embodiment the inventive methods for the production are usedto make agricultural products such as, but not limited to, plantextracts, proteins, amino acids, carbohydrates, fats, oils, polymers,vitamins, and the like.

It is possible that a plant product consists of one or more agriculturalproducts to a large extent.

Methods for Breeding/Methods for Plant Improvement/Methods Plant VarietyProduction

The transgenic plants of the invention may be crossed with similartransgenic plants or with transgenic plants lacking the nucleic acids ofthe invention or with non-transgenic plants, using known methods ofplant breeding, to prepare seeds. Further, the transgenic plant cells orplants of the present invention may comprise, and/or be crossed toanother transgenic plant that comprises one or more exogenous nucleicacids, thus creating a “stack” of transgenes in the plant and/or itsprogeny. The seed is then planted to obtain a crossed fertile transgenicplant comprising the CASAR nucleic acid. The crossed fertile transgenicplant may have the particular expression cassette inherited through afemale parent or through a male parent. The second plant may be aninbred plant. The crossed fertile transgenic may be a hybrid. Alsoincluded within the present invention are seeds of any of these crossedfertile transgenic plants. The seeds of this invention can be harvestedfrom fertile transgenic plants and be used to grow progeny generationsof transformed plants of this invention including hybrid plant linescomprising the exogenous nucleic acid.

Thus, one embodiment of the present invention is a method for breeding afungal resistant plant comprising the steps of

-   (a) crossing a transgenic plant described herein or a plant    obtainable by a method described herein with a second plant;-   (b) obtaining a seed or seeds resulting from the crossing step    described in (a);-   (c) planting said seed or seeds and growing the seed or seeds to    plants; and-   (d) selecting from said plants the plants expressing a CASAR    protein, preferably encoded by    -   (i) an exogenous nucleic acid having at least 60% identity with        SEQ ID NO: 2, 1, 5-12, 13, 15, 17, 19, 21, 23, 25, or 27, a        functional fragment thereof, an orthologue or a paralogue        thereof, or a splice variant thereof;    -   (ii) an exogenous nucleic acid encoding a protein having at        least 60% identity with SEQ ID NO: 3, 14, 16, 18, 20, 22, 24,        26, or 28, or a functional fragment thereof, an orthologue or a        paralogue thereof; preferably the encoded protein confers        enhanced fungal resistance relative to control plants;    -   (iii) an exogenous nucleic acid capable of hybridizing under        stringent conditions with a complementary sequence of any of the        nucleic acids according to (i) or (ii); preferably encoding a        CASAR protein; preferably wherein the nucleic acid molecule        codes for a polypeptide which has essentially identical        properties to the polypeptide described in SEQ ID NO: 3;        preferably the encoded protein confers enhanced fungal        resistance relative to control plants; and/or by    -   (iv) an exogenous nucleic acid encoding the same CASAR protein        as any of the nucleic acids of (i) to (iii) above, but differing        from the nucleic acids of (i) to (iii) above due to the        degeneracy of the genetic code.

Another preferred embodiment is a method for plant improvementcomprising

-   (a) obtaining a transgenic plant by any of the methods of the    present invention;-   (b) combining within one plant cell the genetic material of at least    one plant cell of the plant of (a) with the genetic material of at    least one cell differing in one or more gene from the plant cells of    the plants of (a) or crossing the transgenic plant of (a) with a    second plant;-   (c) obtaining seed from at least one plant generated from the one    plant cell of (b) or the plant of the cross of step (b);-   (d) planting said seeds and growing the seeds to plants; and-   (e) selecting from said plants, plants expressing the nucleic acid    encoding the CASAR protein; and optionally-   (f) producing propagation material from the plants expressing the    nucleic acid encoding the CASAR protein.

The transgenic plants may be selected by known methods as describedabove (e.g., by screening for the presence of one or more markers whichare encoded by plant-expressible genes co-transferred with the CASARgene or screening for the CASAR nucleic acid itself).

According to the present invention, the introduced CASAR nucleic acidmay be maintained in the plant cell stably if it is incorporated into anon-chromosomal autonomous replicon or integrated into the plantchromosomes. Whether present in an extra-chromosomal nonreplicating orreplicating vector construct or a vector construct that is integratedinto a chromosome, the exogenous CASAR nucleic acid preferably residesin a plant expression cassette. A plant expression cassette preferablycontains regulatory sequences capable of driving gene expression inplant cells that are functional linked so that each sequence can fulfillits function, for example, termination of transcription bypolyadenylation signals. Preferred polyadenylation signals are thoseoriginating from Agrobacterium tumefaciens t-DNA such as the gene 3known as octopine synthase of the Ti-plasmid pTiACH5 (Gielen et al.,1984, EMBO J. 3:835) or functional equivalents thereof, but also allother terminators functionally active in plants are suitable. As plantgene expression is very often not limited on transcriptional levels, aplant expression cassette preferably contains other functional linkedsequences like translational enhancers such as the overdrive-sequencecontaining the 5′-untranslated leader sequence from tobacco mosaic virusincreasing the polypeptide per RNA ratio (Gallie et al., 1987, Nucl.Acids Research 15:8693-8711). Examples of plant expression vectorsinclude those detailed in: Becker, D. et al., 1992, New plant binaryvectors with selectable markers located proximal to the left border,Plant Mol. Biol. 20:1195-1197; Bevan, M. W., 1984, Binary Agrobacteriumvectors for plant transformation, Nucl. Acid. Res. 12:8711-8721; andVectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol.1, Engineering and Utilization, eds.: Kung and R. Wu, Academic Press,1993, S. 15-38.

EXAMPLES

The following examples are not intended to limit the scope of the claimsto the invention, but are rather intended to be exemplary of certainembodiments. Any variations in the exemplified methods that occur to theskilled artisan are intended to fall within the scope of the presentinvention.

Example 1 General Methods

The chemical synthesis of oligonucleotides can be affected, for example,in the known fashion using the phosphoamidite method (Voet, Voet, 2ndEdition, Wiley Press New York, pages 896-897). The cloning steps carriedout for the purposes of the present invention such as, for example,restriction cleavages, agarose gel electrophoresis, purification of DNAfragments, transfer of nucleic acids to nitrocellulose and nylonmembranes, linking DNA fragments, transformation of E. coli cells,bacterial cultures, phage multiplication and sequence analysis ofrecombinant DNA, are carried out as described by Sambrook et al. ColdSpring Harbor Laboratory Press (1989), ISBN 0-87969-309-6. Thesequencing of recombinant DNA molecules is carried out with an MWG-Licorlaser fluorescence DNA sequencer following the method of Sanger (Sangeret al., Proc. Natl. Acad. Sci. USA 74, 5463 (1977)).

Example 2 Cloning of Overexpression Vector Constructs

The CASAR cDNA (as shown in SEQ ID NO: 1) was synthesized in a way thatan BamHI restriction site is located in front of the start-ATG and anPstI restriction site downstream of the stop-codon. The synthesized cDNAwas digested using the restriction enzyme PstI (NEB Biolabs), the stickyend of the PstI site was blunted using Mung Bean Nuclease according tomanufactures manual (NEB Biolabs). The blunted fragment was digestedusing the restriction enzyme BamHI and ligated in a HindIII digestedGateway pENTRY-B vector (Invitrogen, Life Technologies, Carlsbad,Calif., USA). The sticky end of the HindIII site was blunted using MungBean Nuclease according to manufactures manual (NEB Biolabs) and theblunted vector was digested with BamHI before the ligation.

To obtain the binary plant transformation vector, a triple LR reaction(Gateway system, Invitrogen, Life Technologies, Carlsbad, Calif., USA)was performed according to manufacturer's protocol by using a pENTRY-Avector containing a parsley ubiquitine promoter, the above describedpENTRY-B vector containing the CASAR gene and a pENTRY-C vectorcontaining the terminator of the cathepsin D inhibitor gene from Solanumtuberosum. As target a binary pDEST vector was used which is composedof: (1) a Spectinomycin/Streptomycin resistance cassette for bacterialselection (2) a pVS1 origin for replication in Agrobacteria (3) a colE-1origin of replication for stable maintenance in E. coli and (4) betweenthe right and left border an AHAS selection under control of apcUbi-promoter (FIG. 2). The recombination reaction was transformed intoE. coli (DH5alpha), mini-prepped and screened by specific restrictiondigestions. A positive clone from each vector construct was sequencedand submitted for soy transformation.

The soybean-expression optimized CASAR cDNA (as shown in SEQ ID NO: 2)was synthesized in a way that an NcoI restriction site is located infront of the start-ATG and an AscI restriction site downstream of thestop-codon. The synthesized cDNAs were digested using the restrictionenzymes NcoI and AscI (NEB Biolabs) and ligated in a NcoI/AscI digestedGateway pENTRY-B vector (Invitrogen, Life Technologies, Carlsbad,Calif., USA) in a way that the full-length fragment is located in sensedirection between the “rust induced mesophyll specific promoter 820”(mesophyll specific promoter, expression induced by Phakopsorapachyrhizi, SEQ ID NO: 4) and the terminator of the cathepsin Dinhibitor gene from Solanum tuberosum (t-StCATHD-pA).

To obtain the binary plant transformation vector, a triple LR reaction(Gateway system, Invitrogen, Life Technologies, Carlsbad, Calif., USA)was performed according to manufacturers protocol by using an emptypENTRY-A vector, the promoter::cDNA::terminator in a pENTRY-B vector,and an empty pENTRY-C vector. As target a binary pDEST vector was usedwhich is composed of: (1) a Spectinomycin/Streptomycin resistancecassette for bacterial selection (2) a pVS1 origin for replication inAgrobacteria (3) a colE-1 origin of replication for stable maintenancein E. coli and (4) between the right and left border an AHAS selectionunder control of a pcUbi-promoter (FIG. 3). The recombination reactionwas transformed into E. coli (DH5alpha), mini-prepped and screened byspecific restriction digestions. A positive clone from each vectorconstruct was sequenced and submitted soy transformation.

Example 3 Soy Transformation

The expression vector constructs (see example 2) were transformed intosoy.

3.1 Sterilization and Germination of Soy Seeds

Virtually any seed of any soy variety can be employed in the method ofthe invention. A variety of soybean cultivar (including Jack, Williams82, Jake, Stoddard and Resnik) is appropriate for soy transformation.Soy seeds were sterilized in a chamber with a chlorine gas produced byadding 3.5 ml 12N HCl drop wise into 100 ml bleach (5.25% sodiumhypochlorite) in a desiccator with a tightly fitting lid. After 24 to 48hours in the chamber, seeds were removed and approximately 18 to 20seeds were plated on solid GM medium with or without 5 μM6-benzyl-aminopurine (BAP) in 100 mm Petri dishes. Seedlings without BAPare more elongated and roots develop, especially secondary and lateralroot formation. BAP strengthens the seedling by forming a shorter andstockier seedling.

Seven-day-old seedlings grown in the light (>100 μEinstein/m²s) at 25°C. were used for explant material for the three-explant types. At thistime, the seed coat was split, and the epicotyl with the unifoliateleaves have grown to, at minimum, the length of the cotyledons. Theepicotyl should be at least 0.5 cm to avoid the cotyledonary-node tissue(since soycultivars and seed lots may vary in the developmental time adescription of the germination stage is more accurate than a specificgermination time).

For inoculation of entire seedlings, see Method A (example 3.3.1 and3.3.2) or leaf explants, see Method B (example 3.3.3).

For method C (see example 3.3.4), the hypocotyl and one and a half orpart of both cotyledons were removed from each seedling. The seedlingswere then placed on propagation media for 2 to 4 weeks. The seedlingsproduce several branched shoots to obtain explants from. The majority ofthe explants originated from the plantlet growing from the apical bud.These explants were preferably used as target tissue.

3.2—Growth and Preparation of Agrobacterium Culture

Agrobacterium cultures were prepared by streaking Agrobacterium (e.g.,A. tumefaciens or A. rhizogenes) carrying the desired binary vector(e.g. H. Klee. R. Horsch and S. Rogers 1987 Agrobacterium-Mediated PlantTransformation and its further Applications to Plant Biology; AnnualReview of Plant Physiology Vol. 38: 467-486) onto solid YEP growthmedium (YEP media: 10 g yeast extract, 10 g Bacto Peptone, 5 g NaCl,Adjust pH to 7.0, and bring final volume to 1 liter with H2O, for YEPagar plates add 20 g Agar, autoclave) and incubating at 25° C. untilcolonies appeared (about 2 days). Depending on the selectable markergenes present on the Ti or Ri plasmid, the binary vector, and thebacterial chromosomes, different selection compounds were be used for A.tumefaciens and A. rhizogenes selection in the YEP solid and liquidmedia. Various Agrobacterium strains can be used for the transformationmethod.

After approximately two days, a single colony (with a sterile toothpick)was picked and 50 ml of liquid YEP was inoculated with antibiotics andshaken at 175 rpm (25° C.) until an OD₆₀₀ between 0.8-1.0 is reached(approximately 2 d). Working glycerol stocks (15%) for transformationare prepared and one-ml of Agrobacterium stock aliquoted into 1.5 mlEppendorf tubes then stored at −80° C.

The day before explant inoculation, 200 ml of YEP were inoculated with 5μl to 3 ml of working Agrobacterium stock in a 500 ml Erlenmeyer flask.The flask was shaken overnight at 25° C. until the OD₆₀₀ was between 0.8and 1.0. Before preparing the soy explants, the Agrobacteria werepelleted by centrifugation for 10 min at 5,500×g at 20° C. The pelletwas resuspended in liquid CCM to the desired density (OD₆₀₀ 0.5-0.8) andplaced at room temperature at least 30 min before use.

3.3—Explant Preparation and Co-Cultivation (Inoculation) 3.3.1 Method A:Explant Preparation on the Day of Transformation.

Seedlings at this time had elongated epicotyls from at least 0.5 cm butgenerally between 0.5 and 2 cm. Elongated epicotyls up to 4 cm in lengthhad been successfully employed. Explants were then prepared with: i)with or without some roots, ii) with a partial, one or both cotyledons,all preformed leaves were removed including apical meristem, and thenode located at the first set of leaves was injured with several cutsusing a sharp scalpel.

This cutting at the node not only induced Agrobacterium infection butalso distributed the axillary meristem cells and damaged pre-formedshoots. After wounding and preparation, the explants were set aside in aPetri dish and subsequently co-cultivated with the liquidCCM/Agrobacterium mixture for 30 minutes. The explants were then removedfrom the liquid medium and plated on top of a sterile filter paper on15×100 mm Petri plates with solid co-cultivation medium. The woundedtarget tissues were placed such that they are in direct contact with themedium.

3.3.2 Modified Method A: Epicotyl Explant Preparation

Soyepicotyl segments prepared from 4 to 8 d old seedlings were used asexplants for regeneration and transformation. Seeds of soya cv.L00106CN, 93-41131 and Jack were germinated in 1/10 MS salts or asimilar composition medium with or without cytokinins for 4 to 8 d.Epicotyl explants were prepared by removing the cotyledonary node andstem node from the stem section. The epicotyl was cut into 2 to 5segments. Especially preferred are segments attached to the primary orhigher node comprising axillary meristematic tissue.

The explants were used for Agrobacterium infection. Agrobacterium AGL1harboring a plasmid with the gene of interest (GOI) and the AHAS, bar ordsdA selectable marker gene was cultured in LB medium with appropriateantibiotics overnight, harvested and resuspended in a inoculation mediumwith acetosyringone. Freshly prepared epicotyl segments were soaked inthe Agrobacterium suspension for 30 to 60 min and then the explants wereblotted dry on sterile filter papers. The inoculated explants were thencultured on a co-culture medium with L-cysteine and TTD and otherchemicals such as acetosyringone for increasing T-DNA delivery for 2 to4 d. The infected epicotyl explants were then placed on a shootinduction medium with selection agents such as imazapyr (for AHAS gene),glufosinate (for bar gene), or D-serine (for dsdA gene). The regeneratedshoots were sub-cultured on elongation medium with the selective agent.

For regeneration of transgenic plants the segments were then cultured ona medium with cytokinins such as BAP, TDZ and/or Kinetin for shootinduction. After 4 to 8 weeks, the cultured tissues were transferred toa medium with lower concentration of cytokinin for shoot elongation.Elongated shoots were transferred to a medium with auxin for rooting andplant development. Multiple shoots were regenerated.

Many stable transformed sectors showing strong cDNA expression wererecovered. Soybean plants were regenerated from epicotyl explants.Efficient T-DNA delivery and stable transformed sectors weredemonstrated.

3.3.3 Method B: Leaf Explants

For the preparation of the leaf explant the cotyledon was removed fromthe hypocotyl. The cotyledons were separated from one another and theepicotyl is removed. The primary leaves, which consist of the lamina,the petiole, and the stipules, were removed from the epicotyl bycarefully cutting at the base of the stipules such that the axillarymeristems were included on the explant. To wound the explant as well asto stimulate de novo shoot formation, any pre-formed shoots were removedand the area between the stipules was cut with a sharp scalpel 3 to 5times.

The explants are either completely immersed or the wounded petiole enddipped into the Agrobacterium suspension immediately after explantpreparation. After inoculation, the explants are blotted onto sterilefilter paper to remove excess Agrobacterium culture and place explantswith the wounded side in contact with a round 7 cm Whatman paperoverlaying the solid CCM medium (see above). This filter paper preventsA. tumefaciens overgrowth on the soy-explants. Wrap five plates withParafilm™ “M” (American National Can, Chicago, Ill., USA) and incubatefor three to five days in the dark or light at 25° C.

3.3.4 Method C: Propagated Axillary Meristem

For the preparation of the propagated axillary meristem explantpropagated 3-4 week-old plantlets were used. Axillary meristem explantscan be pre-pared from the first to the fourth node. An average of threeto four explants could be obtained from each seedling. The explants wereprepared from plantlets by cutting 0.5 to 1.0 cm below the axillary nodeon the internode and removing the petiole and leaf from the explant. Thetip where the axillary meristems lie was cut with a scalpel to induce denovo shoot growth and allow access of target cells to the Agrobacterium.Therefore, a 0.5 cm explant included the stem and a bud.

Once cut, the explants were immediately placed in the Agrobacteriumsuspension for 20 to 30 minutes. After inoculation, the explants wereblotted onto sterile filter paper to remove excess Agrobacterium culturethen placed almost completely immersed in solid CCM or on top of a round7 cm filter paper overlaying the solid CCM, depending on theAgrobacterium strain. This filter paper prevents Agrobacteriumovergrowth on the soy-explants. Plates were wrapped with Parafilm™“M”(American National Can, Chicago, Ill., USA) and incubated for two tothree days in the dark at 25° C.

3.4—Shoot Induction

After 3 to 5 days co-cultivation in the dark at 25° C., the explantswere rinsed in liquid SIM medium (for SIM, see Olhoft et al., A novelAgrobacterium rhizogenes-mediated transformation method of soy usingprimary-node explants from seedlings, In Vitro Cell. Dev. Biol.—Plant(2007) 43:536-549) or Modwash medium (1×B5 major salts, 1×B5 minorsalts, 1×MSIII iron, 3% Sucrose, 1×B5 vitamins, 30 mM MES, 350 mg/LTimentin™ pH 5.6, WO 2005/121345) to remove excess Agrobacterium andblotted dry on sterile filter paper (to prevent damage especially on thelamina) before placing on the solid SIM medium. The approximately 5explants (Method A) or 10 to 20 (Methods B and C) explants were placedsuch that the target tissue was in direct contact with the medium.During the first 2 weeks, the explants could be cultured with or withoutselective medium. Preferably, explants were transferred onto SIM withoutselection for one week.

For leaf explants (Method B), the explant should be placed into themedium such that it is perpendicular to the surface of the medium withthe petiole imbedded into the medium and the lamina out of the medium.

For propagated axillary meristem (Method C), the explant was placed intothe medium such that it was parallel to the surface of the medium(basipetal) with the explant partially embedded into the medium.

Wrap plates with Scotch 394 venting tape (3M, St. Paul, Minn., USA) wereplaced in a growth chamber for two weeks with a temperature averaging25° C. under 18 h light/6 h dark cycle at 70-100 μE/m²s. The explantsremained on the SIM medium with or without selection until de novo shootgrowth occurred at the target area (e.g., axillary meristems at thefirst node above the epicotyl). Transfers to fresh medium can occurduring this time. Explants were transferred from the SIM with or withoutselection to SIM with selection after about one week. At this time,there was considerable de novo shoot development at the base of thepetiole of the leaf explants in a variety of SIM (Method B), at theprimary node for seedling explants (Method A), and at the axillary nodesof propagated explants (Method C).

Preferably, all shoots formed before transformation were removed up to 2weeks after co-cultivation to stimulate new growth from the meristems.This helped to reduce chimerism in the primary transformant and increaseamplification of transgenic meristematic cells. During this time theexplant may or may not be cut into smaller pieces (i.e. detaching thenode from the explant by cutting the epicotyl).

3.5—Shoot Elongation

After 2 to 4 weeks (or until a mass of shoots was formed) on SIM medium(preferably with selection), the explants were transferred to SEM medium(shoot elongation medium, see Olhoft et al., A novel Agrobacteriumrhizogenes-mediated transformation method of soy using primary-nodeexplants from seedlings, In Vitro Cell. Dev. Biol. Plant (2007)43:536-549) that stimulates shoot elongation of the shoot primordia.This medium may or may not contain a selection compound.

After every 2 to 3 weeks, the explants were transferred to fresh SEMmedium (preferably containing selection) after carefully removing deadtissue. The explants should hold together and not fragment into piecesand retain somewhat healthy. The explants were continued to betransferred until the explant dies or shoots elongate. Elongatedshoots >3 cm were removed and placed into RM medium for about 1 week(Method A and B), or about 2 to 4 weeks depending on the cultivar(Method C) at which time roots began to form. In the case of explantswith roots, they were transferred directly into soil. Rooted shoots weretransferred to soil and hardened in a growth chamber for 2 to 3 weeksbefore transferring to the greenhouse. Regenerated plants obtained usingthis method were fertile and produced on average 500 seeds per plant.

After 5 days of co-cultivation with Agrobacterium tumefaciens transientexpression of the gene of interest (GOI) was widespread on the seedlingaxillary meristem explants especially in the regions wounding duringexplant preparation (Method A). Explants were placed into shootinduction medium without selection to see how the primary-node respondsto shoot induction and regeneration. Thus far, greater than 70% of theexplants were formed new shoots at this region. Expression of the GOIwas stable after 14 days on SIM, implying integration of the T-DNA intothe soy genome. In addition, preliminary experiments resulted in theformation of cDNA expressing shoots forming after 3 weeks on SIM.

For Method C, the average regeneration time of a soy plantlet using thepropagated axillary meristem protocol was 14 weeks from explantinoculation. Therefore, this method has a quick regeneration time thatleads to fertile, healthy soy plants.

Example 4 Pathogen Assay 4.1. Growth of Plants

10 T1 plants per event were potted and grown for 3-4 weeks in thephytochamber (16 h-day-und 8 h-night-Rhythm at a temperature of 16 and22° C. and a humidity of 75%) till the first 2 trifoliate leaves werefully expanded.

4.2 Inoculation

The plants were inoculated with P. pachyrhizi.

In order to obtain appropriate spore material for the inoculation,soybean leaves which had been infected with rust 15-20 days ago, weretaken 2-3 days before the inoculation and transferred to agar plates (1%agar in H2O). The leaves were placed with their upper side onto theagar, which allowed the fungus to grow through the tissue and to producevery young spores. For the inoculation solution, the spores were knockedoff the leaves and were added to a Tween-H2O solution. The counting ofspores was performed under a light microscope by means of a Thomacounting chamber. For the inoculation of the plants, the sporesuspension was added into a compressed-air operated spray flask andapplied uniformly onto the plants or the leaves until the leaf surfaceis well moisturized. For macroscopic assays we used a spore density of1-5×10⁵ spores/ml. For the microscopy, a density of >5×10⁵ spores/ml isused. The inoculated plants were placed for 24 hours in a greenhousechamber with an average of 22° C. and >90% of air humidity. Thefollowing cultivation was performed in a chamber with an average of 25°C. and 70% of air humidity.

Example 5 Microscopical Screening

For the evaluation of the pathogen development, the inoculated leaves ofplants were stained with aniline blue 48 hours after infection.

The aniline blue staining serves for the detection of fluorescentsubstances. During the defense reactions in host interactions andnon-host interactions, substances such as phenols, callose or ligninaccumulated or were produced and were incorporated at the cell walleither locally in papillae or in the whole cell (hypersensitivereaction, HR). Complexes were formed in association with aniline blue,which lead e.g. in the case of callose to yellow fluorescence. The leafmaterial was transferred to falcon tubes or dishes containing destainingsolution II (ethanol/acetic acid 6/1) and was incubated in a water bathat 90° C. for 10-15 minutes. The destaining solution II was removedimmediately thereafter, and the leaves were washed 2× with water. Forthe staining, the leaves were incubated for 1.5-2 hours in stainingsolution II (0.05% aniline blue=methyl blue, 0.067 M di-potassiumhydrogen phosphate) and analyzed by microscopy immediately thereafter.

The different interaction types were evaluated (counted) by microscopy.An Olympus UV microscope BX61 (incident light) and a UV Longpath filter(excitation: 375/15, Beam splitter: 405 LP) are used. After aniline bluestaining, the spores appeared blue under UV light. The papillae could berecognized beneath the fungal appressorium by a green/yellow staining.The hypersensitive reaction (HR) was characterized by a whole cellfluorescence.

Example 6 Evaluating the Susceptibility to Soybean Rust

The progression of the soybean rust disease was scored by the estimationof the diseased area (area which was covered by sporulating uredinia) onthe backside (abaxial side) of the leaf. Additionally the yellowing ofthe leaf was taken into account (for scheme see FIG. 1).

At all 43 T₁ soybean plants (5 independent events, 7-10 transgenicplants each; expression of transgene checked by RT-PCR) expressing theCASAR protein were inoculated with spores of Phakopsora pachyrhizi Themacroscopic disease symptoms caused by P. pachyrhizi on the inoculatedsoybean plants were scored 14 days after inoculation.

The average of the percentage of the leaf area showing fungal coloniesor strong yellowing/browning on all leaves was considered as diseasedleaf area. At all 43 soybean T₁ plants expressing CASAR (expressionchecked by RT-PCR) were evaluated in parallel to non-transgenic controlplants of the same variety. Non-transgenic soy plants grown in parallelto the transgenic plants were used as control. The average of thediseased leaf area is shown in FIG. 7 for plants exogenously expressingCASAR compared with wildtype plants. Overexpression of CASAR reduces thediseased leaf area in comparison to non-transgenic control plants by44.5% in average over all 5 independent events tested. This data clearlyindicates that the in-planta expression of the CASAR expression vectorconstruct lead to a lower disease scoring of transgenic plants comparedto non-transgenic controls. So, the expression of CASAR (as shown in SEQID NO: 1) in soybean significantly (p<0.001) increases the resistance ofsoybean against soybean rust.

1. A method for increasing fungal resistance in a plant, a plant part,or a plant cell wherein the method comprises the step of increasing theexpression and/or activity of a CASAR protein in the plant, plant part,or plant cell in comparison to a wild type plant, wild type plant partor wild type plant cell, wherein the fungal resistance is resistanceagainst rust.
 2. The method of claim 1, wherein the CASAR proteincomprises an amino acid sequence having at least 60% identity with SEQID NO: 3, 14, 16, 18, 20, 22, 24, 26, or 28, or a functional fragmentthereof, an orthologue or a paralogue thereof.
 3. The method of claim 1,wherein the CASAR protein is encoded by (i) an exogenous nucleic acidcomprising a nucleic acid sequence having at least 60% identity with SEQID NO: 2, 1, 5-12, 13, 15, 17, 19, 21, 23, 25, or 27, or a functionalfragment thereof, an orthologue or a paralogue thereof, or a splicevariant thereof; (ii) an exogenous nucleic acid encoding a proteincomprising an amino acid sequence having at least 60% identity with SEQID NO: 3, 14, 16, 18, 20, 22, 24, 26, or 28, or a functional fragmentthereof, an orthologue or a paralogue thereof; (iii) an exogenousnucleic acid capable of hybridizing under stringent conditions with acomplementary sequence of any of the nucleic acids according to (i) or(ii); and/or by (iv) an exogenous nucleic acid encoding the same CASARprotein as any of the nucleic acids of (i) to (iii) above, but differingfrom the nucleic acids of (i) to (iii) above due to the degeneracy ofthe genetic code.
 4. The method of claim 1, comprising: (a) stablytransforming a plant cell with an expression cassette comprising (i) anexogenous nucleic acid comprising a nucleic acid sequence having atleast 60% identity with SEQ ID NO: 2, 1, 5-12, 13, 15, 17, 19, 21, 23,25, or 27, or a functional fragment thereof, an orthologue or aparalogue thereof, or a splice variant thereof; (ii) an exogenousnucleic acid encoding a protein comprising an amino acid sequence havingat least 60% identity with SEQ ID NO: 3, 14, 16, 18, 20, 22, 24, 26, or28, or a functional fragment thereof, an orthologue or a paraloguethereof; (iii) an exogenous nucleic acid capable of hybridizing understringent conditions with a complementary sequence of any of the nucleicacids according to (i) or (ii), and/or (iv) an exogenous nucleic acidencoding the same CASAR protein as any of the nucleic acids of (i) to(iii) above, but differing from the nucleic acids of (i) to (iii) abovedue to the degeneracy of the genetic code, in functional linkage with arust-induced promoter; (b) regenerating the plant from the plant cell;and (c) expressing said exogenous nucleic acid.
 5. A recombinant vectorconstruct comprising: (a) (i) a nucleic acid comprising a nucleic acidsequence having at least 60% identity with SEQ ID NO: 2, 1, 5-12, 13,15, 17, 19, 21, 23, 25, or 27, or a functional fragment thereof, anorthologue or a paralogue thereof, or a splice variant thereof; (ii) anucleic acid encoding a protein comprising an amino acid sequence havingat least 60% identity with SEQ ID NO: 3, 14, 16, 18, 20, 22, 24, 26, or28, or a functional fragment thereof, an orthologue or a paraloguethereof; (iii) a nucleic acid capable of hybridizing under stringentconditions with a complementary sequence of any of the nucleic acidsaccording to (i) or (ii), and/or (iv) a nucleic acid encoding the sameCASAR protein as any of the nucleic acids of (i) to (iii) above, butdiffering from the nucleic acids of (i) to (iii) above due to thedegeneracy of the genetic code, operably linked with (b) a rust-inducedpromoter and (c) a transcription termination sequence.
 6. The method ofclaim 4, wherein the promoter is a mesophyll-specific promoter or anepidermis-specific promoter.
 7. A transgenic soy plant, transgenic soyplant part, or transgenic soy plant cell transformed with a recombinantvector construct comprising: (a) (i) a nucleic acid comprising a nucleicacid sequence having at least 60% identity with SEQ ID NO: 2, 1, 5-12,13, 15, 17, 19, 21, 23, 25, or 27, or a functional fragment thereof, anorthologue or a paralogue thereof, or a splice variant thereof; (ii) anucleic acid encoding a protein comprising an amino acid sequence havingat least 60% identity with SEQ ID NO: 3, 14, 16, 18, 20, 22, 24, 26, or28, or a functional fragment thereof, an orthologue or a paraloguethereof; (iii) a nucleic acid capable of hybridizing under stringentconditions with a complementary sequence of any of the nucleic acidsaccording to (i) or (ii), and/or (iv) a nucleic acid encoding the sameCASAR protein as any of the nucleic acids of (i) to (iii) above, butdiffering from the nucleic acids of (i) to (iii) above due to thedegeneracy of the genetic code, operably linked with (b) a promoter and(c) a transcription termination sequence.
 8. A method for the productionof a transgenic plant, transgenic plant part, or transgenic plant cellhaving increased resistance against rust, comprising (a) introducing arecombinant vector construct comprising: (i) a nucleic acid comprising anucleic acid sequence having at least 60% identity with SEQ ID NO: 2, 1,5-12, 13, 15, 17, 19, 21, 23, 25, or 27, or a functional fragmentthereof, an orthologue or a paralogue thereof, or a splice variantthereof; (ii) a nucleic acid encoding a protein comprising an amino acidsequence having at least 60% identity with SEQ ID NO: 3, 14, 16, 18, 20,22, 24, 26, or 28, or a functional fragment thereof, an orthologue or aparalogue thereof; (iii) a nucleic acid capable of hybridizing understringent conditions with a complementary sequence of any of the nucleicacids according to (i) or (ii), and/or (iv) a nucleic acid encoding thesame CASAR protein as any of the nucleic acids of (i) to (iii) above,but differing from the nucleic acids of (i) to (iii) above due to thedegeneracy of the genetic code, operably linked with a promoter and atranscription termination sequence into a plant, a plant part, or aplant cell; (b) generating a transgenic plant, transgenic plant part, ortransgenic plant cell from the plant, plant part or plant cell; and (c)expressing the CASAR protein encoded by (i) the exogenous nucleic acidcomprising a nucleic acid sequence having at least 60% identity with SEQID NO: 2, 1, 5-12, 13, 15, 17, 19, 21, 23, 25, or 27, a functionalfragment thereof, an orthologue or a paralogue thereof, or a splicevariant thereof; (ii) the exogenous nucleic acid encoding a proteincomprising an amino acid sequence having at least 60% identity with SEQID NO: 3, 14, 16, 18, 20, 22, 24, 26, or 28, or a functional fragmentthereof, an orthologue or a paralogue thereof; (iii) the exogenousnucleic acid capable of hybridizing under stringent conditions with acomplementary sequence of any of the nucleic acids according to (i) or(ii); and/or by (iv) the exogenous nucleic acid encoding the same CASARprotein as any of the nucleic acids of (i) to (iii) above, but differingfrom the nucleic acids of (i) to (iii) above due to the degeneracy ofthe genetic code.
 9. The method of claim 8, further comprising the stepof harvesting the seeds of the transgenic plant and planting the seedsand growing the seeds to plants, wherein the grown plants comprise (i)the exogenous nucleic acid comprising a nucleic acid sequence having atleast 60% identity with SEQ ID NO: 2, 1, 5-12, 13, 15, 17, 19, 21, 23,25, or 27, a functional fragment thereof, an orthologue or a paraloguethereof, or a splice variant thereof; (ii) the exogenous nucleic acidencoding a protein comprising an amino acid sequence having at least 60%identity with SEQ ID NO: 3, 14, 16, 18, 20, 22, 24, 26, or 28, or afunctional fragment thereof, an orthologue or a paralogue thereof; (iii)the exogenous nucleic acid capable of hybridizing under stringentconditions with a complementary sequence of any of the nucleic acidsaccording to (i) or (ii); and/or (iv) the exogenous nucleic acidencoding the same CASAR protein as any of the nucleic acids of (i) to(iii) above, but differing from the nucleic acids of (i) to (iii) abovedue to the degeneracy of the genetic code.
 10. (canceled)
 11. Aharvestable part of a transgenic plant of claim
 7. 12. A product derivedfrom the plant of claim
 7. 13. A method for the production of a productcomprising: a) growing the plant of claim 7 and b) producing saidproduct from or by the plant and/or part, of the plant.
 14. The methodof claim 13, wherein said product is produced from or by the harvestableparts of the plant.
 15. The method of claim 13, wherein the product ismeal or oil.
 16. The method claim 1, wherein the fungal resistance is aresistance against soybean rust.
 17. The method of claim 16, wherein theresistance against soybean rust is resistance against Phakopsorameibomiae and/or Phakopsora pachyrhizi.
 18. The method of claim 1,wherein the plant is selected from the group consisting of beans, soy,pea, clover, kudzu, lucerne, lentils, lupins, vetches, groundnut, rice,wheat, barley, arabidopsis, lentil, banana, canola, cotton, potato,corn, sugar cane, alfalfa, and sugar beet.
 19. A method for breeding arust resistant plant comprising: (a) crossing the plant of claim 7 witha second plant; (b) obtaining seed from the cross of step (a); (c)planting said seeds and growing the seeds to plants; and (d) selectingfrom said plants expressing an CASAR protein encoded by (i) theexogenous nucleic acid comprising a nucleic acid sequence having atleast 60% identity with SEQ ID NO: 2, 1, 5-12, 13, 15, 17, 19, 21, 23,25, or 27, a functional fragment thereof, an orthologue or a paraloguethereof, or a splice variant thereof; (ii) the exogenous nucleic acidencoding a protein comprising an amino acid sequence having at least 60%identity with SEQ ID NO: 3, 14, 16, 18, 20, 22, 24, 26, or 28, or afunctional fragment thereof, an orthologue or a paralogue thereof; (iii)the exogenous nucleic acid capable of hybridizing under stringentconditions with a complementary sequence of any of the nucleic acidsaccording to (i) or (ii); and/or by (iv) the exogenous nucleic acidencoding the same CASAR protein as any of the nucleic acids of (i) to(iii) above, but differing from the nucleic acids of (i) to (iii) abovedue to the degeneracy of the genetic code.
 20. The harvestable part ofclaim 11, wherein the harvestable part is a transgenic seed of thetransgenic plant.
 21. The product of claim 12, wherein the product issoybean meal or soy oil.